Lipid Nanoparticle Composition

Abstract

The present disclosure relates to compositions of RNA encapsulated in lipid nanoparticles and methods for producing the same. The compositions can be used to deliver mRNA to a subject. The lipid nanoparticles have improved properties for delivering biologically active agents, such as RNA.

Description

[Technical Field]

[0001] Related application data This application claims priority to U.S. Provisional Patent Application No. 63 / 476,217, entitled "Lipid nanoparticle composition," filed December 20, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure relates to compositions of mRNA encapsulated in lipid nanoparticles and methods for producing the same. The compositions can be used to deliver mRNA to a subject. [Background technology]

[0004] Nucleic acid-based therapies have shown great potential in a variety of therapeutic applications. However, the delivery of polynucleotides, such as messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotides, plasmids, and DNA, presents numerous challenges. Free nucleic acids, such as RNA, are subject to rapid enzymatic degradation and generally do not persist throughout the body. Furthermore, due to their negative charge, nucleic acids may not be able to effectively cross cellular barriers and enter necessary intracellular compartments, e.g., for translation, or otherwise exert their effects. This is particularly true for mRNA, which can be a very large molecule with a high negative charge density. Furthermore, mRNA is highly susceptible to degradation by 5' exonucleases, 3' exonucleases, and endonucleases, making it an inherently unstable molecule.

[0005] Therefore, lipid nanoparticles (LNPs) have been used to formulate nucleic acids to protect them from degradation and improve cellular uptake and intracellular delivery. LNPs are typically formed from ionizable cationic lipids and other lipid components, such as neutral lipids, sterols (e.g., cholesterol), and PEGylated lipids. Ionizable cationic lipids are amphiphilic molecules with a lipophilic region containing one or more hydrocarbon groups and a hydrophilic region containing at least one positively charged or ionizable polar head group. These cationic lipids ionize at the appropriate pH, forming positively charged complexes with nucleic acids, facilitating their passage through the cell membrane and into the cytoplasm.

[0006] The first approved siRNA therapeutic, Onpattro (patisiran), entered the market just a few years ago for the treatment of hereditary amyloidogenic transthyretin (TTR) amyloidosis. Patisiran's therapeutic efficacy relies on siRNA-mediated TTR gene silencing, preventing the production of mutant proteins and, at the very least, disease progression. Efficient delivery of siRNA relies on LNP technology. More recently, nucleic acid vaccines, including those against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the ongoing severe coronavirus disease 2019 (COVID-19) pandemic, have been used to treat and prevent a variety of diseases. mRNA vaccines rely on the delivery of mRNA into the cytoplasm of host cells, where it is transcribed into antigenic proteins and triggers the generation of an immune response. The large size and negative charge of mRNA hinder cellular uptake, again necessitating LNP for proper delivery. Summary of the Invention [Problem to be solved by the invention]

[0007] The method of preparing LNP can include solvent mixing, homogenization and microfluidics.However, these methods often produce a heterogeneous mixture, which does not always provide efficient delivery of mRNA cargo.Therefore, there is a need for a method that can be used to produce a concentrated population of LNP suitable for delivering mRNA to subject, for example, with low heterogeneity. [Means for solving the problem]

[0008] The present disclosure is based, at least in part, on the experimental finding that the effectiveness of a composition comprising lipid nanoparticles can be improved by removing unencapsulated mRNA from the composition. Accordingly, the present application provides a method for preparing an enriched population of lipid nanoparticles, the method comprising contacting a composition comprising lipid nanoparticles and mRNA with an anion exchanger under conditions such that the anion exchanger binds to the unencapsulated mRNA, and collecting the effluent to obtain an enriched population of lipid nanoparticles. In one embodiment, the unencapsulated mRNA comprises free mRNA, mRNA bound to the surface of the lipid nanoparticles, and / or partially exposed mRNA.

[0009] In one embodiment, the composition comprising lipid nanoparticles has an ion concentration of 5 mM to 50 mM. In one embodiment, the composition comprising lipid nanoparticles and RNA comprises a salt at a concentration of 100 mM or less. In one embodiment, the composition comprising lipid nanoparticles and RNA comprises a salt at a concentration of 40 mM or less. In one embodiment, the composition comprising lipid nanoparticles has a conductivity of less than 15 mS / cm.

[0010] In one embodiment, the composition comprising lipid nanoparticles further comprises a buffer selected from citrate buffer, Bis-Tris, histidine, acetate buffer, phosphate buffer, Tris buffer, and / or combinations thereof, hi one embodiment, the buffer is Bis-Tris buffer, histidine buffer, or Tris buffer.

[0011] In one embodiment, the buffer is a citrate buffer or a Tris buffer. In one embodiment, the buffer is citrate. In one embodiment, the buffer is Tris. In one embodiment, the composition comprises one or more additional components selected from a sugar, a polymer, and a surfactant.

[0012] In one embodiment, the lipid nanoparticle comprises a lipid component and mRNA. In one embodiment, the lipid component comprises a lipid selected from the group consisting of an ionizable lipid, a neutral lipid, a lipid bound to a hydrophilic polymer, a structured lipid, and combinations thereof. In one embodiment, the lipid component comprises an ionizable lipid, a neutral lipid, a lipid bound to a hydrophilic polymer, and a structured lipid. In one embodiment, the lipid component comprises an ionizable lipid, a neutral lipid, a PEGylated lipid, and a structured lipid.

[0013] In one embodiment, the LNP comprises an ionizable lipid and one or more of a neutral lipid, a PEGylated lipid, and a structured lipid.

[0014] In one embodiment, the ionizable lipid is 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), 1,2-Dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 2-Hexyldecanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester (ALC-0315), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA or MC3), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), and 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester.

[0015] In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

[0016] In one embodiment, the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, optionally PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

[0017] In one embodiment, the structured lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and α-tocopherol, hi one embodiment, the structured lipid is cholesterol and / or campesterol.

[0018] In one embodiment, the LNP comprises a lipid component comprising about 25 mol% to about 60 mol% ionizable lipid, about 2 mol% to about 25 mol% neutral lipid, about 18.5 mol% to about 60 mol% structural lipid, and about 0.2 mol% to about 10 mol% PEGylated lipid.

[0019] In one embodiment, the mRNA is a self-amplifying mRNA (sa-mRNA) or a conventional mRNA (cRNA). In one embodiment, the mRNA is a self-amplifying mRNA. In one embodiment, the mRNA is a cRNA.

[0020] In one embodiment, the mRNA is greater than 500 nt in length. In one embodiment, the RNA is between 500 nt and 20,000 nt in length.

[0021] In one embodiment, the anion exchanger is an anion exchange resin or an anion exchange membrane. In one embodiment, the anion exchanger is an anion exchange membrane selected from Mustang® Q, Sartobind® Q, Chromasorb®, Capto® Q, Q Sepharose Fast Flow (QSFF), Poros® Q, Fractogel® EMD, Natrix® Q, or Eshmuno® Q membranes. In one embodiment, the anion exchanger is a Mustang® Q membrane.

[0022] In one embodiment, the method further comprises eluting the unencapsulated mRNA from the anion exchange material.

[0023] In one embodiment, the lipid nanoparticles have a diameter of about 30 nm to about 160 nm. In one embodiment, the lipid nanoparticles have a diameter of about 60 nm to about 130 nm. In one embodiment, the lipid nanoparticles have a diameter of about 70 nm to about 120 nm. In one embodiment, the lipid nanoparticles have a diameter of about 80 nm to about 120 nm. In one embodiment, the lipid nanoparticles have a diameter of about 70 nm to about 100 nm.

[0024] The present disclosure also provides a composition comprising an enriched population of lipid nanoparticles produced by the methods described herein. In one embodiment, at least 90% of the RNA is encapsulated within the LNPs. In one embodiment, the percentage of encapsulated RNA is measured using anion exchange chromatography. In one embodiment, the percentage of encapsulated RNA is measured using a Ribogreen assay. In one embodiment, the percentage of encapsulated RNA is measured by determining the amount of RNA present in the composition before and after contact with an anion exchanger.

[0025] The present disclosure also provides a concentrated lipid nanoparticle composition comprising: (i) a plurality of lipid nanoparticles, each LNP comprising an ionizable lipid, a neutral lipid, a PEGylated lipid, and a structured lipid; and (ii) mRNA, wherein at least 90% of the mRNA is encapsulated within the LNP. In one embodiment, at least about 95% of the mRNA is encapsulated within the LNP. In one embodiment, at least about 97% of the mRNA is encapsulated within the LNP.

[0026] The present disclosure also provides a pharmaceutical composition comprising an enriched population of lipid nanoparticles produced by the methods described herein and a pharmaceutically acceptable carrier.

[0027] The present disclosure also provides a pharmaceutical composition comprising an enriched population of lipid nanoparticles described herein and a pharmaceutically acceptable carrier.

[0028] In one example, the potency (e.g., in vitro potency) of the composition is at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold greater than the untreated composition, where treating includes contacting with an anion exchanger. In one example, the potency (e.g., in vitro potency) of the composition is at least 4-fold greater than the untreated composition.

[0029] The present disclosure also provides a method of delivering mRNA to a mammalian cell, comprising administering to a subject a pharmaceutical composition described herein, thereby contacting the cell with the lipid nanoparticles and delivering the mRNA to the cell. In one example, the cell is a cell of a human subject.

[0030] The present disclosure also provides a method of producing a polypeptide of interest in a mammalian cell, comprising contacting the cell with a pharmaceutical composition described herein.

[0031] The present disclosure also provides a method of treating a disease, disorder, or condition in a subject in need of such treatment, comprising administering to the subject a pharmaceutical composition described herein, thereby treating the disease, disorder, or condition. In one example, the disease, disorder, or condition is selected from the group consisting of a rare disease, an infectious disease, a cancer, a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

[0032] The present disclosure also provides for the use of a pharmaceutical composition described herein in the manufacture of a medicament for the treatment of a disease, disorder, or condition, in one example, the disease, disorder, or condition is selected from the group consisting of a rare disease, an infectious disease, a cancer, a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

[0033] The present disclosure also provides a vaccine comprising a composition described herein, a concentrated lipid nanoparticle composition described herein, or a pharmaceutical composition described herein. In one example, the vaccine is selected from a tumor vaccine, an influenza vaccine, and a SARS vaccine, including SARS-CoV-2.

[0034] The present disclosure provides a method for generating an immune response in a subject, the method comprising administering to the subject a composition comprising an enriched population of LNPs, wherein the LNPs comprise an ionizable lipid, a phospholipid, a PEG-lipid, and a structural lipid, and at least 90% of the LNPs comprise RNA encapsulated within the LNPs.

[0035] In one example, the potency (e.g., in vitro potency) of the composition is at least 1.5-fold, 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold greater than the untreated composition, where treating includes contacting with an anion exchanger. In one example, the potency (e.g., in vitro potency) of the composition is at least 4-fold greater than the untreated composition.

[0036] In one example, the amount of RNA administered to a subject is about 10%, 20%, 30%, 30%, or 50% of the RNA that would be administered to a subject in an untreated composition, where treating includes contacting with an anion exchanger. In one example, the amount of RNA administered to a subject is about 10% of the RNA that would be administered to a subject if the composition had not been filtered using an anion exchange filter.

[0037] In one example, the amount of RNA administered to a subject is about 10 μg or less. [Brief explanation of the drawings]

[0038] [Figure 1] The in vitro activity and potency (probability of successful transfection per mass unit of RNA) of treated and untreated LNPs as measured by AF4-MALS and fluorescence-activated cell sorting (FACS) are shown. [Figure 2A] 1 shows the in vitro expression levels of H5 in filtered and unfiltered LNP. [Figure 2B] 1 shows the in vitro expression levels of N1 in filtered and unfiltered LNP. [Figure 3A] Shown are total IgG responses quantified by ELISA from mice immunized with treated and untreated LNPs 21 days after the first vaccination. [Figure 3B] Shown are total IgG responses quantified by ELISA from mice immunized with treated and untreated LNPs 42 days after the first vaccination. [Figure 4] Hemagglutinin titers from mice immunized with treated and untreated LNP are shown 42 days after the first vaccination. [Figure 5] Pseudotype virus neutralization titers of mice immunized with treated and untreated LNPs are shown 42 days after the first vaccination. [Figure 6A] Microneutralization titers from mice immunized with treated and untreated LNPs in a short-form microneutralization assay 42 days after the first vaccination are shown. [Figure 6B] Microneutralization titers from mice immunized with treated and untreated LNPs in a long-form microneutralization assay 42 days after the first vaccination are shown. [Figure 7] 1 shows antibody responses of mice immunized with treated and untreated LNPs 42 days after the first vaccination, as assessed by ELLA. [Figure 8A] Dose comparison of mice immunized with 0.01 μg of self-replicating RNA is shown. [Figure 8B] Dose comparison of mice immunized with 0.1 μg of self-replicating RNA is shown. [Figure 9A] Figure 1 is a graph showing the net % of HA-specific CD4+ response. Cytokines assayed were IFNγ, IL5 and / or IL13, and IL2 and / or TNFα. [Figure 9B] Figure 1 is a graph showing the net % of NA-specific CD4+ responses. Cytokines assayed were IFNγ, IL5 and / or IL13, and IL2 and / or TNFα. [Figure 9C] Figure 1 is a graph showing the net % of HA-specific CD8+ responses. Cytokines assayed were IFNγ, IL5 and / or IL13, and IL2 and / or TNFα. [Figure 9D] Figure 1 is a graph showing the net % of NA-specific CD8+ responses. Cytokines assayed were IFNγ, IL5 and / or IL13, and IL2 and / or TNFα. [Figure 10A] Dose comparison of mice immunized with 1 μg of self-replicating RNA, 0.1 μg of self-replicating RNA, 0.01 μg of self-replicating RNA, or 0.001 μg of self-replicating RNA is shown. The graph shows the net % of HA-specific CD4+ response. [Figure 10B] Dose comparison of mice immunized with 1 μg of self-replicating RNA, 0.1 μg of self-replicating RNA, 0.01 μg of self-replicating RNA, or 0.001 μg of self-replicating RNA is shown. The graph shows the net % of NA-specific CD4+ response. [Figure 10C] Dose comparison of mice immunized with 1 μg of self-replicating RNA, 0.1 μg of self-replicating RNA, 0.01 μg of self-replicating RNA, or 0.001 μg of self-replicating RNA is shown. The graph shows the net % of HA-specific CD8+ response. [Figure 10D] Dose comparison of mice immunized with 1 μg of self-replicating RNA, 0.1 μg of self-replicating RNA, 0.01 μg of self-replicating RNA, or 0.001 μg of self-replicating RNA is shown. The graph shows the net % of NA-specific CD8+ response.

[0039] Sequence table legend [Table 1]

[0040] Although the sequence listing refers to DNA sequences, the disclosure of this application should be understood to include their RNA equivalents and their complements, unless the context clearly dictates otherwise. DETAILED DESCRIPTION OF THE INVENTION

[0041] General Throughout this specification, unless expressly stated otherwise or the context requires otherwise, references to a single step, a single composition, a group of steps, or a group of compositions shall be interpreted as encompassing one and more (i.e., one or more) of those steps, compositions, steps, or compositions.

[0042] Those skilled in the art will recognize that the present disclosure is susceptible to variations and modifications other than those specifically described, and it is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any combination or any two or more of such steps or features.

[0043] The present disclosure is not to be limited in scope by the specific examples described herein, as such specific examples are intended for illustrative purposes only. Functionally equivalent objects, compositions, and methods are clearly within the scope of the present disclosure.

[0044] Any example of the present disclosure should be construed as applicable mutatis mutandis to any other example of the present disclosure, unless specifically stated otherwise, i.e., any specific example of the present disclosure can be combined with any other specific example of the present disclosure (except where mutually exclusive).

[0045] Any example of the present disclosure disclosing a specific feature or group of features, or a method or method step, is deemed to provide explicit support for disclaiming the specific feature or group of features, or the method or method step.

[0046] Unless specifically defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., of synthetic organic chemistry, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0047] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. These techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current revisions), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and J.E. Coligan et al. al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all current revisions), and other sources.

[0048] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing clear support for both meanings or either meaning.

[0049] The terms "from" and "to," when used to indicate a range, should be understood to mean that the range includes the recited lower and upper limits. For example, "x is an integer from 0 to 6" should be understood to include the situation where x is absent (x is 0), the situation where x is 6, and each integer value therebetween, i.e., x is 1, 2, 3, 4, or 5.

[0050] As used herein, "about" means the numerical value itself and / or within 10% of the stated numerical value. For example, "about 5%" means 5 and / or any number or range within the range of 4.5 to 5.5, e.g., 4.5 to 4.96, 4.81 to 5.35, etc. In one embodiment, "about" means the numerical value itself and / or within 5% of the stated numerical value.

[0051] It will be understood that throughout this specification the word "comprise" or variations such as "comprises" or "comprising" imply the inclusion of a stated component, element or step, or group of components, elements or steps, but not the exclusion of any other component, element or step, or group of components, elements or steps.

[0052] As used herein, the term "derived from" should be understood to indicate that the identified component may be obtained from a particular source, but is not necessarily obtained directly from that source. Similarly, the term "based on" should be understood to indicate that the identified end product is derived from or used in a particular source, but is not necessarily derived from or used directly from that source.

[0053] Selected Definitions As used herein, the term "chromatography" refers to any type of technique that separates a product of interest (e.g., LNPs containing encapsulated RNA) from contaminants and / or other components in a preparation.

[0054] As used herein, the term "flow-through" refers to a product separation technique intended to allow a preparation containing a desired product to flow through a material. In one example, the desired product flows through the material, while undesired substances bind to the material. In one example, the material is an anion exchanger. As used herein, the term "effluent" refers to material that is not adsorbed onto the anion exchanger and is eluted with the mobile phase (e.g., water). In some examples, effluent and flow-through are used interchangeably.

[0055] As used herein, the terms "contaminant" or "impurity" are used interchangeably herein and refer to any foreign or unwanted molecule, including biological macromolecules such as DNA, RNA, and one or more additives that may be present in a sample containing a product of interest to be separated from one or more of the foreign or unwanted molecules. Additionally, such contaminants may include any reagents used in steps that may occur prior to the separation process. In one example, a contaminant may include aggregates of phospholipids (e.g., DSPC) and structural lipids (e.g., cholesterol). In one example, an impurity includes unencapsulated RNA. In one example, an impurity includes partially encapsulated RNA. In one example, the method described herein is intended to selectively remove unencapsulated or exposed RNA from a sample containing a product of interest.

[0056] As used herein, the term "substantially pure," when used in the context of an LNP population, refers to an LNP population in which at least 90% of the LNPs comprise encapsulated RNA, as measured, for example, by anion exchange chromatography or Ribogreen assay. In one example, the percentage of encapsulated RNA is measured using a Ribogreen assay. In one example, the percentage of encapsulated RNA is measured using anion exchange chromatography. In one example, a substantially pure LNP population has an encapsulation rate of about 95%, or about 97%, or about 99%.

[0057] As used herein, the term "polynucleotide" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, including DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may be in the form of short hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. mRNA includes sa-mRNA and cRNA. Polynucleotides include synthetic, natural, and non-natural polynucleotides containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties to a reference polynucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, the term encompasses polynucleotides containing known analogs of natural nucleotides that have similar binding properties as the reference polynucleotide. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). In one example, the polynucleotide is mRNA.

[0058] As used herein, the terms "disease," "disorder," or "condition" refer to a disruption of or interference with normal function and include, but are not limited to, any particular condition, a disease or disorder.

[0059] As used herein, a subject "at risk" of developing a disease, disorder, or condition may or may not have detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms prior to treatment according to the present disclosure. "At risk" means that the subject has one or more risk factors, which are measurable parameters that correlate with development of a disease or condition, known in the art and / or described herein.

[0060] As used herein, the terms "treating," "treat," or "treatment" include administering an RNA or composition described herein to thereby reduce or eliminate at least one symptom of a particular disease or condition.

[0061] As used herein, the terms "preventing," "prevent," or "prevention" include providing prophylaxis against the occurrence or recurrence of a particular disease or condition in an individual. An individual may be susceptible to or at risk of developing a disease, but has not yet been diagnosed with the disease.

[0062] As used herein, the phrase "delaying the progression of" includes reducing or delaying the progression of a disease or condition, and / or at least one symptom of a disease or condition, in an individual.

[0063] The term "pharmaceutical composition" refers to a formulation containing a therapeutically active agent, preferably in association with a pharmaceutically acceptable carrier, diluent, and / or excipient. Such pharmaceutical compositions are useful for treating, preventing, or reducing the severity of a disease or disorder by administration to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[0064] An "effective amount" refers to at least an amount effective, at a dosage and for a period of time necessary to achieve a desired result. For example, the desired result can be a therapeutic or prophylactic result. In some embodiments of the present disclosure, the term "effective amount" or "therapeutically effective amount" of a therapeutic mRNA is an amount sufficient to produce a desired effect, such as increased or inhibited expression of a target sequence compared to normal expression levels detected in the absence of that mRNA. Suitable assays for measuring expression of a target gene or target sequence include examination of protein or mRNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of appropriate reporter proteins, and phenotypic assays. An effective amount may vary depending on the disease or condition being treated or on changing factors, and may further vary depending on the body weight, age, racial background, sex, health and / or physical condition, and other factors related to the mammal being treated. Typically, an effective amount falls within a relatively broad range (e.g., a "dosage" range) that can be determined by routine testing and experimentation by a medical professional. Therefore, this term should not be interpreted to limit the present disclosure to a specific amount (e.g., weight or mRNA number). An effective amount can be provided in one or more administrations. For example, an effective amount can be administered in a single dose, or in a dose that is repeated once or several times during a treatment period.

[0065] A "therapeutically effective amount" is at least the minimum concentration required to produce a measurable improvement in a particular disease or condition. The therapeutically effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the mRNA of the present disclosure to elicit a desired response in an individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the mRNA are outweighed by the therapeutically beneficial effects.

[0066] A "prophylactically effective amount" shall be understood to mean an amount of mRNA of the present disclosure sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a disease or disorder described herein.

[0067] As used herein, the term "subject" shall be taken to mean any animal (e.g., mammal), including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0068] As used herein, the term "mammal" includes humans and both domestic animals, such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife.

[0069] As used herein, "zeta potential" is the electrokinetic potential of a lipid, for example, a lipid in a lipid nanoparticle composition.

[0070] Anion Exchanger The present inventors have discovered that enhanced efficacy of compositions containing LNPs can be achieved by a method including the use of an anion exchanger. According to the present disclosure, an enriched population of lipid nanoparticles can be prepared by contacting a composition containing lipid nanoparticles and mRNA with an anion exchanger under conditions such that the ion exchanger binds to unencapsulated (e.g., exposed) mRNA. For example, mRNA protected from the environment by being encapsulated within lipid nanoparticles does not bind to the anion exchanger and remains in the unbound fraction. The unbound fraction is then separated from the anion exchanger to obtain an enriched population of LNPs. In one example, the flow-through is collected to obtain an enriched population of lipid nanoparticles. In one example, unencapsulated RNA bound to the ion exchanger is separated from the composition to obtain an enriched population of LNPs.

[0071] In this context, the term "anion exchanger" is used to encompass any means for carrying out an anion exchange step. For example, the term "anion exchanger" can refer to a matrix or solid support to which one or more positively charged ligands, such as quaternary amino groups, are attached. Thus, the term "anion exchanger" specifically includes, but is not limited to, anion exchange resins, matrices, absorbers, membranes (including membrane adsorbers), and the like. Anion exchangers are known to those skilled in the art. Anion exchangers suitable for use in the methods described herein include, but are not limited to, commercially available Mustang® Q, Sartobind® Q, Chromasorb®, Capto® Q, Q Sepharose Fast Flow (QSFF), Poros® Q, Fractogel® EMD (e.g., Fractogel® EMD TMAE, Fraetogel® HMD TAE highcap, and Fractogel® EMD DEAE), Natrix® Q, Eshmuno® Q, DEAE cellulose, QAE SEPHADEX™, and the like. In one embodiment, the "anion exchanger" is an anion exchange membrane. In one embodiment, the anion exchange membrane is a Mustang® Q membrane, such as a Mustang® Q filter.

[0072] Typically, anion exchange membranes have a nominal pore size of 0.1 to 100 μm. For reference, Sartobind® Q (Sartorius AG) is a strong anion exchange membrane with a nominal pore size of 3 to 5 μm, available commercially in either single-layer or multilayer formats, and Mustang® Q (Pall Corporation) is a strong anion exchange membrane with a nominal pore size of 0.8 μm, also available commercially in either single-layer or multilayer formats. The "nominal" pore size rating refers to the membrane's ability to retain a majority of particles at 60 to 98% of the rated pore size. In one embodiment, the nominal pore size is about 0.1 to 5 μm. In one embodiment, the nominal pore size is about 0.1 to 3 μm. In one embodiment, the nominal pore size is about 0.1 to 1 μm, e.g., 0.8 μm.

[0073] In an embodiment where the anion exchanger is a membrane, the membrane can be made from a variety of suitable materials. In one embodiment, the membrane is polyethersulfone (PES) (e.g., from Millipore or PALL Corp.). In one embodiment, the membrane is regenerated cellulose (RC) (e.g., from Sartorius or Pierce). In one embodiment, the anion exchanger is a Q membrane, which is a positively charged membrane and an anion exchanger that includes a quaternary amine. For example, a Q membrane is functionalized with a quaternary ammonium (R-CH2-N(CH3)3). In some embodiments, the anion exchanger is a diethylamine group (R-CH2NH + The membrane is a weakly basic anion exchanger functionalized with (C2H5)2). In one embodiment, the membrane is a weakly basic anion exchanger comprising diethylaminoethyl (DEAE) cellulose. In one embodiment, the membrane comprises quaternary amine functional groups. In one embodiment, the membrane is a polyethersulfone (PES)-based membrane (e.g., Mustang Q membrane) with a cross-linked polymer coating of quaternary amine functional groups.

[0074] In embodiments where the anion exchanger is a membrane, the anion exchanger may comprise a single layer of membrane, or may comprise more than one layer of membrane, e.g., 2, 3, 4, 6, 8, 10, 12, 14, or 16 or more layers of membrane. In one embodiment, the anion exchanger comprises four layers of membrane. In one embodiment, the anion exchanger comprises 16 layers of membrane.

[0075] In embodiments where the anion exchanger is a membrane, the anion exchanger may comprise a flat sheet, a pleated sheet, or a unipleat® cartridge. In one embodiment, the anion exchanger is a flat sheet. In one embodiment, the anion exchanger is pleated. In one embodiment, the membrane is a unipleat® cartridge.

[0076] In one embodiment, the anion exchanger, e.g., an anion exchange membrane, is contained in a device used in centrifugation (e.g., a spin column), or in a device used in a vacuum system (e.g., a vacuum filter holder), or in a device used in pressure filtration (e.g., a syringe filter), or in a device used in chromatography (e.g., a column). In one embodiment, the anion exchanger is contained in a syringe filter.

[0077] In one example, the anion exchanger is housed in a column that can be run on either a standard or custom chromatography system, such as an AKTA™ Explorer (GE Healthcare), equipped with a pressure gauge, a sensor, and a pump and pump controller. In this example, the anion exchanger is installed downstream of the pressure gauge. In one example, a pH detector and a conductivity detector are installed downstream of the anion exchanger. In one example, before installing the anion exchanger, the system is thoroughly flushed with water, followed by an equilibration buffer. In one example, the system with the membrane is flushed with an equilibration buffer, for example, until the pH and conductivity outlets of the solution match the specifications of the equilibration buffer (e.g., approximately 5 membrane volumes) and a stable baseline is observed. In one example, a composition comprising lipid nanoparticles and RNA is buffer-exchanged into an equilibration buffer before contacting the anion exchanger. In one example, the feed material is loaded by a pump at an appropriate pH (i.e., a pH at which unencapsulated mRNA has a negative charge and LNPs have a neutral or positive charge) and appropriate conductivity. The operating backpressure, as well as changes in pH and conductivity, are recorded during operation. Finally, the membrane effluent containing the enriched LNP population is collected. In one example, the membrane effluent containing the enriched LNP population is collected when the ultraviolet (UV) absorbance trace at 280 nm (other wavelengths, such as 260 nm or 254 nm, can be used) is 0.2 absorbance units above baseline. Pool collection is stopped when the UV trace at 280 nm falls below 0.2 absorbance units, and the RNA concentration of a sample from the pooled membrane effluent fraction is analyzed. In one example, the effluent containing the enriched LNP population is collected without monitoring the absorbance trace. In one example, after the contacting step, the anion exchanger is washed with an equilibration buffer. The step recovery is typically calculated using the total RNA loaded and the total RNA in the membrane effluent. In one example, the anion exchange membrane is disposable. In one example, the anion exchange membrane can be reused by treating it with a wash buffer (such as a high-salt buffer) and / or a regeneration buffer.

[0078] In one example, the effluent is collected and the anion exchanger is contacted with a high salt buffer to elute the compound (e.g., unencapsulated RNA bound to the anion exchanger). In one example, the high salt buffer comprises at least 200 mM salt, 300 mM salt, 400 mM salt, 500 mM salt, or 1 M salt. In some examples, the salt is NaCl, although any suitable salt may be used.

[0079] In one embodiment, the composition subjected to the enrichment method described herein is a lipid nanoparticle composition prepared using techniques known to those skilled in the art. As will be understood by those skilled in the art, the composition may include lipid nanoparticles, RNA-encapsulated LNPs, and unencapsulated RNA. As used herein, "unencapsulated RNA" is broadly defined to include free RNA, RNA bound to the surface of LNPs, and partially encapsulated RNA. In other words, RNA is considered unencapsulated if it is fully or partially exposed to the surrounding environment. The composition may also include one or more optional components, such as ethanol, buffer, salt, etc.

[0080] Optionally, the LNP composition is subjected to at least one purification step before contacting it with the anion exchanger.

[0081] In one embodiment, the LNP composition is desalted before contacting with the anion exchanger. In one embodiment, the LNP composition is subjected to a buffer exchange step before contacting with the anion exchanger. The pH of the buffer is such that unencapsulated RNA binds to the anion exchanger, while encapsulated RNA does not substantially bind to the anion exchanger. In one embodiment, the pH of the LNP-containing composition is adjusted to a pH below 10. In one embodiment, the pH of the LNP-containing composition is adjusted to a pH of about 6 to about 8. In one embodiment, the pH of the load material is adjusted to about 7 to 8, or about 7.5. In one embodiment, the pH of the LNP-containing composition is adjusted to, for example, a pH of about 6 to about 8, and the conductivity of the load material is adjusted to about 50 mS / cm or less, depending on the pH, before the LNP-containing composition is contacted with the anion exchanger. In one example, the pH of the LNP-containing composition is adjusted, e.g., to about 6.5 to about 7.5, and the conductivity of the load material is adjusted to about 50 mS / cm or less, depending on the pH, before contacting the LNP-containing composition with an anion exchanger. In one example, the pH of the LNP-containing composition is adjusted, e.g., to about 6 to about 8, and the ionic concentration of the load material is adjusted to about 50 mS / cm or less, depending on the pH, before contacting the LNP-containing composition with an anion exchanger. In one example, the pH of the LNP-containing composition is adjusted, e.g., to about 6.5 to about 7.5, and the ionic concentration of the load material is adjusted to about 50 mM or less, depending on the pH, before contacting the LNP-containing composition with an anion exchanger. In one example, the conductivity of the load material is adjusted to about 50 mS / cm or less, e.g., about 40 mS / cm or less, about 30 mS / cm or less, about 20 mS / cm or less, or about 10 mS / cm or less. In one example, the conductivity of the load material is adjusted to about 20 mS / cm or less or about 10 mS / cm or less, depending on the pH. In one example, the ionic concentration of the load material is adjusted to about 50 mM or less, e.g., about 40 mM or less, about 30 mM or less, about 20 mM or less, or about 10 mM or less. In one example, the ionic concentration of the load material is adjusted to about 40 mM or less or about 36 mM, depending on the pH. Under these conditions, unencapsulated RNA has a negative charge and therefore electrostatically binds to the positive functional groups of the anion exchanger.This is because the unencapsulated RNA (negative) and the membrane (positive) have opposite charges. Without wishing to be bound by theory, the negative charge of the encapsulated RNA (i.e., the RNA contained inside the lipid nanoparticle) is shielded from the anion exchanger, so that under pH and conductivity conditions that induce charges with minimal ionic shielding, the encapsulated RNA does not bind to the membrane, while the unencapsulated RNA does, thereby allowing the encapsulated RNA to "elute" or pass through the matrix and be recovered in the effluent.

[0082] In one embodiment, a method is provided for purifying LNP-encapsulated mRNA from a composition comprising the LNP-encapsulated mRNA and at least one contaminant, the method comprising: (a) passing the composition through an anion exchanger, wherein the contaminant and the anion exchanger have opposite charges, under operating conditions comprising a buffer having a pH and conductivity selected to cause the membrane to bind to the contaminant; and b) recovering the purified mRNA encapsulated in the LNP from the effluent.

[0083] In one embodiment, a method is provided for purifying LNP-encapsulated mRNA from a composition comprising the LNP-encapsulated mRNA and at least one contaminant, the method comprising: (a) passing the composition through an anion exchanger, where the contaminant and the anion exchanger have opposite charges, under operating conditions comprising a buffer having a pH of 7-10 and a conductivity of about 100 mS / cm or less that binds the membrane to the contaminant; and b) recovering the purified LNP-encapsulated mRNA from the effluent.

[0084] With regard to analytical assays, RNA content can be determined using techniques known to those skilled in the art, for example, by absorbance at 260 nm using a spectrophotometer, or using a fluorescence-based assay such as Ribogreen.

[0085] buffer solution In some embodiments, the methods provided herein include various buffers, including equilibration buffers, loading buffers, and wash buffers. The buffers can include various components. In some embodiments, the buffers include one or more of Tris, Bis-Tris, Bis-Tris-propane, imidazole, citrate, methylmalonic acid, acetic acid, ethanolamine, diethanolamine, triethanolamine (TEA), and sodium phosphate. Additionally, any buffer can be adjusted up or down in pH by adding an acid or base (e.g., acetic acid, citric acid, HEPES, hydrochloric acid, phosphoric acid, sodium hydroxide, TRIS, or other such acidic and basic buffers) to reach the appropriate pH. Any buffer system can also be adjusted up or down in conductivity using purified water, water for injection (WFI), sodium acetate, sodium chloride, potassium phosphate, or other such low and high salt buffers to reach the appropriate conductivity.

[0086] In some embodiments, the ionic strength of the equilibration buffer, loading buffer, and wash buffer can be high or low. In some embodiments, the ionic strength of the equilibration buffer and loading buffer can be low. In some embodiments, the buffer includes a salt, e.g., a chloride salt such as NaCl. In some embodiments, the salt concentration can be 0-0.3M. In one embodiment, the salt concentration is 0 mM, 10 mM, 25 mM, 50 mM, 100 mM, or 150 mM. In one embodiment, the salt concentration is about 100 mM. In one embodiment, the salt concentration is about 0 mM.

[0087] In some embodiments, the equilibration buffer, loading buffer, and wash buffer may also contain other components, such as sugars or polymers. In some embodiments, the equilibration buffer, loading buffer, and wash buffer may also contain sugars. Suitable sugars include, but are not limited to, disaccharides (e.g., glucose, sucrose, or trehalose, or combinations thereof). In some embodiments, the total sugar concentration ranges from 0 w / w% to about 30 w / w%. For example, the sugar concentration ranges from 0 w / w% to about 25 w / w% (e.g., about 0-25 w / w%, 0-20 w / w%, 0-15 w / w%, 0-10 w / w%, about 5 w / w%, about 8 w / w%, about 10 w / w%, about 15 w / w%, about 20 w / w%, or about 25 w / w%).

[0088] In some embodiments, the equilibration buffer, loading buffer, and wash buffer may also contain a polymer. Suitable polymers include, but are not limited to, poloxamers (Pluronic®), poloxamines (Tetronic®), polyoxyethylene glycol sorbitan alkyl esters (polysorbates), and polyvinylpyrrolidone (PVP). As will be appreciated by those skilled in the art, the components of the buffer should not disrupt the LNP or should only minimally disrupt the LNP. In some embodiments, the polymer is present at a concentration ranging from about 0.1% w / v to about 3% w / v, or from about 0.1% w / w to about 3% w / w. For example, the polymer is present at a concentration ranging from about 0.1% w / v to about 3% w / v, or from about 0.1% w / w to about 3% w / w.

[0089] lipid nanoparticles The method of the present disclosure can be used to prepare a concentrated population of LNPs. In some embodiments, LNPs (e.g., concentrated populations of LNPs) can be used to deliver mRNA to a subject. The inventors of the present application have found that concentrated populations of LNPs have increased efficacy compared to unfiltered LNPs.

[0090] As used herein, the term "lipid nanoparticle" or "LNP" is understood to refer to a lipid-based particle having at least one nanometer-order dimension (e.g., 1-1,000 nm). In one embodiment, the term "lipid nanoparticle" includes any lipid-based particle, including, but not limited to, liposomes or vesicles in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; unilamellar or multiple; multilamellar), micelle-like lipid nanoparticles with a non-aqueous core, and solid lipid nanoparticles. In one embodiment, lipid nanoparticles or LNPs may have a structure comprising a single monolayer or bilayer of lipids encapsulating a solid phase. In one embodiment, lipid nanoparticles or LNPs have no aqueous or other liquid phase within their interior. In one embodiment, lipid nanoparticles or LNPs have no substantial aqueous or other liquid phase within their interior. In one embodiment, LNPs are formed by combining an aqueous composition comprising RNA with an organic composition comprising lipids.

[0091] In examples, the LNPs are formulated into compositions for delivery of mRNA to a desired target, such as a cell, tissue, organ, tumor, or the like.

[0092] In one embodiment, the lipid nanoparticle comprises a lipid component and mRNA. In one embodiment, the lipid component comprises a lipid selected from the group consisting of an ionizable lipid, a neutral lipid, a lipid bound to a hydrophilic polymer, a structured lipid, and combinations thereof. In one embodiment, the lipid component comprises an ionizable lipid, a neutral lipid, a lipid bound to a hydrophilic polymer, and a structured lipid. In one embodiment, the lipid component comprises an ionizable lipid, a neutral lipid, a PEGylated lipid, and a structured lipid.

[0093] LNPs generally comprise ionizable and / or cationic lipids, as well as one or more of neutral lipids, charged lipids, sterols, and PEGylated lipids.

[0094] The terms "cationic lipid," "ionizable cationic lipid," "cationic lipid compound," "ionizable cationic lipid compound," or similar terms refer to lipid compounds that can bear a positive charge. In one embodiment, the ionizable cationic lipids disclosed herein contain one or more nitrogen-containing groups that can bear a positive charge. Because these compounds are ionizable, they can exist in either a positively charged or neutral form, depending on the pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticles under various pH conditions. In one embodiment, the cationic lipids have a positive charge at a pH of less than about 7, less than about 6, or less than about 5.

[0095] The LNPs may include cationic and / or ionizable lipids, such as cationic and / or ionizable lipids that include cyclic or acyclic amines. Such additional cationic and / or ionizable lipids may be selected from the non-limiting group consisting of: 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), 1,2-Dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (also known as heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) (SM-102), 2-Hexyldecanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester (also known as ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) (ALC-0315), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA or MC3), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), and 8-[(2-Hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester.

[0096] In one example, the phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (also known as LKY750).

[0097] The term "charged lipid" refers to any of a number of lipid species that exist in positively or negatively charged form regardless of pH within a useful physiological range (e.g., about pH 3 to about pH 9). Non-limiting examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylmositols, sterol hemisuccinates, dialkyltrimethylammonium propanes (including DOTAP and DOTMA), dialkyldimethylaminopropanes, ethylphosphocholines, and dimethylaminoethanecarbamoylsterols.

[0098] In an embodiment, the LNP additionally comprises one or more of a PEG lipid, a sterol-structured lipid, and / or a neutral lipid.

[0099] In one example, the present disclosure provides an LNP comprising a neutral lipid. The term "neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. Neutral lipids may also be referred to as "zwitterionic lipids." At physiological pH, such lipids include, but are not limited to, phosphatidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and phosphatidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM).Neutral or zwitterionic lipids suitable for use in the present disclosure will be apparent to those of skill in the art, and examples include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. The lipids can be saturated or unsaturated. In one embodiment, the neutral lipid is DSPC.

[0100] In one embodiment, the present disclosure provides LNPs comprising structured lipids, including, but not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol, and combinations thereof.

[0101] In one embodiment, the structured lipid is a sterol. In an embodiment, the structured lipid is cholesterol. In another embodiment, the structured lipid is campesterol.

[0102] In one embodiment, the present disclosure provides LNPs comprising a lipid conjugated to a hydrophilic polymer, such as polyethylene glycol (PEG). In one embodiment, the present disclosure provides LNPs comprising a PEGylated lipid. It will be apparent to those skilled in the art that PEGylated lipid refers to a lipid modified with polyethylene glycol. Exemplary PEGylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, PEG lipids include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof. In one embodiment, the average molecular weight of the PEG is 5,000 or less, 4,000 or less, 3,000 or less, 2,000 or less, or 1,000 or less. In one embodiment, the average molecular weight of the PEG is about 2,000. In one embodiment, the PEG lipid comprises DMG-PEG2000. In one embodiment, the PEGylated lipid is not a hydroxyl-PEG-lipid. In one embodiment, the PEGylated lipid is a methoxy-PEG lipid.

[0103] In embodiments, the LNPs comprise an ionizable lipid and / or a cationic lipid, a neutral lipid, a sterol such as cholesterol, and a PEGylated lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG (e.g., DMG-PEG2000) and / or the structural lipid may be cholesterol. In one embodiment, the LNPs comprise an ionizable lipid and / or a cationic lipid, DSPC, cholesterol, and DMG-PEG2000. In other embodiments, the cationic lipid and / or ionizable lipid may be LKY750.

[0104] The LNPs are formulated with the mRNA to be delivered to a subject.

[0105] In some embodiments, the lipid component of the LNP formulation comprises about 25 mol% to about 60 mol% cationic lipids and / or ionizable lipid compounds, about 2 mol% to about 25 mol% phospholipids (neutral lipids), about 18.5 mol% to about 60 mol% structural lipids (sterols), and about 0.2 mol% to about 10 mol% PEGylated lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the LNP formulation comprises about 30 mol% to about 50 mol% cationic lipids and / or ionizable lipid compounds, about 5 mol% to about 20 mol% phospholipids, about 30 mol% to about 55 mol% structural lipids, and about 1 mol% to about 5 mol% PEGylated lipids. In particular embodiments, the lipid component comprises about 40 mol% cationic lipids and / or ionizable lipids, about 10 mol% phospholipids, about 48 mol% structural lipids, and about 2.0 mol% PEGylated lipids.

[0106] In the examples, the LNP is about 30 nm to about 160 nm, about 40 nm to about 160 nm, about 50 nm to about 160 nm, about 60 nm to about 160 nm, about 70 nm to about 160 nm, about 50 nm m ~ about 140nm, about 60nm - about 130nm, about 70nm - about 120nm, about 80nm - about 120nm, about 90nm - about 120nm, about 70 - about 110nm, about 80nm - about 1 The lipid nanoparticles have an average diameter of 10 nm, or about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, or 160 nm. In one embodiment, the lipid nanoparticles have a diameter of about 70 nm to about 130 nm, about 70 nm to about 120 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, or about 70 nm to about 90 nm. In one embodiment, the lipid nanoparticles have a diameter of about 70 nm to about 120 nm. In one embodiment, the average diameter of the LNPs is about 80 nm to about 120 nm. In one embodiment, the lipid nanoparticles have a diameter of about 70 nm to about 100 nm.

[0107] The diameter of the LNPs can be measured by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), or other methods such as those known in the art.

[0108] In some embodiments, the particle size of the LNPs can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the LNPs. A small polydispersity index, e.g., less than 0.3 or less than 0.2, generally indicates a narrow particle size distribution. The LNP compositions described herein can have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the LNP composition can be about 0 to about 0.20 or 0.05 to 0.20.

[0109] Suitable methods for producing lipid nanoparticles will be apparent to those skilled in the art and / or are described herein. LNPs comprising an mRNA component and at least one lipid component can be formed using mixing processes such as microfluidics, including, for example, herringbone micromixing and T-junction mixing of two fluid streams (one containing the mRNA, typically in aqueous solution, and the other with various essential lipid components, typically in ethanol).

[0110] In one example, LNPs can be prepared by combining cationic and / or ionizable lipids, phospholipids (e.g., DOPE or DSPC, available from commercial sources including Avanti Polar Lipids, Alabaster, AL), PEGylated lipids (e.g., 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as PEG-DMG, available from commercial sources including Avanti Polar Lipids, Alabaster, AL), and structural lipids / sterols (e.g., cholesterol, available from commercial sources including Sigma-Aldrich) at a concentration of, for example, about 50 mM in ethanol. The solution should be refrigerated during storage, e.g., at -20°C. Various lipids can be combined to obtain the desired molar ratio and diluted with water and ethanol to the desired final lipid concentration, e.g., about 5.5 mM to about 25 mM.

[0111] LNP compositions containing mRNA are prepared by combining the lipid solution described above with a solution containing mRNA, for example, in a lipid component to mRNA weight:weight ratio of about 5:1 to about 50:1 (as shown in the Examples). The lipid solution can be rapidly injected into the mRNA solution at a flow rate of about 3 ml / min to about 18 ml / min using the NanoAssemblr microfluidic system to produce a suspension with a water to ethanol ratio of about 1:1 to about 4:1, or about 2:1 to about 4:1.

[0112] For LNP compositions containing mRNA, a 1.0 mg / ml concentration of mRNA solution in deionized water can be diluted with 50 mM sodium citrate buffer at pH 3-6 to form a stock solution.

[0113] The above LNP preparation methods are believed to result in nanoprecipitation and particle formation. Alternative processes, including but not limited to T-junction and direct injection, can be used to achieve the same nanoprecipitation and form lipid nanoparticle compositions that can be used in the methods described herein.

[0114] The lipid nanoparticle composition can be further processed before or after use in the methods described herein. Suitable techniques include, but are not limited to, dialysis or tangential flow filtration (TFF) to achieve ethanol removal and / or buffer exchange. For example, the formulation can be dialyzed twice against a buffer, such as phosphate-buffered saline (PBS) at pH 7.4, at a volume 200 times the primary product volume, using a Slide-A-Lyzer cassette with a 10 kD molecular weight cutoff (Thermo Fisher Scientific Inc., Rockford, IL). The first dialysis can be performed at room temperature for 3 hours. The second dialysis can be performed overnight at 4°C. In another example, the LNP composition can be further processed by diluting it 10-fold into a first buffer, such as 50 mM citrate buffer at pH 6, and subjecting it to tangential flow filtration (TFF) using a 300,000 molecular weight cutoff membrane (mPES) until concentrated to the original volume. The first buffer can then be replaced with a second buffer using diafiltration with 10 times the volume of the second buffer (e.g., a second buffer containing 20 mM Tris buffer, pH 7.5, 80 mM sodium chloride, and 3% sucrose). The LNP solution can be concentrated to a volume of 5-10 mL, filtered using a 0.2 micron filter, aliquoted into vials, and frozen at 1°C / min using, for example, a Corning® CoolCell® LX Cell Freezing Container until the sample reaches -80°C. Samples can be stored at -80°C until needed.

[0115] messenger RNA The methods of the present disclosure can be used to prepare enriched populations of lipid nanoparticles containing encapsulated mRNA.

[0116] As used herein, the term "messenger RNA" (also referred to as mRNA) refers to any polynucleotide that encodes a polypeptide of interest and that can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide of interest. mRNA may or may not be chemically modified. mRNA of the present disclosure encompasses non-self-replicating mRNA (also referred to as conventional mRNA (cRNA)) and self-replicating RNA (sa-mRNA). In one example, the mRNA is sa-mRNA. In one example, the mRNA is cRNA.

[0117] Typically, cRNA comprises, in 5' to 3' order, a 5' cap structure, a 5'-UTR, a nucleotide sequence encoding a polypeptide of interest, a 3'-UTR, and a tailing sequence (e.g., a polyadenylation signal or polyA tail). cRNAs of the present disclosure may further comprise a translational internal ribosome entry site (e.g., a Kozak consensus sequence or IRES). In some examples, cRNAs may also comprise chain-terminating nucleotides and / or stem-loops.

[0118] As used herein, the term "self-replicating RNA" refers to a construct based on an RNA virus engineered to enable the expression of heterologous RNA and proteins. Self-replicating RNAs can also be referred to as replicons. Self-replicating RNAs can be amplified within a host cell, resulting in the expression of a desired gene product within the host cell. For example, the present disclosure provides a monocistronic self-replicating RNA. The sa-mRNA of the present disclosure contains one or more characteristics of a cRNA, but further contains a nucleotide sequence encoding a nonstructural protein (NSP), enabling the sa-mRNA to direct its own replication. The nonstructural protein includes at least one gene selected from the group consisting of a viral replicase (or viral polymerase), a viral protease, a viral helicase, and other nonstructural viral proteins. Those skilled in the art will appreciate that in one example, the self-replicating RNA can be based on the genomic RNA of an RNA virus. Because the RNA should be a positive (+) strand, it can be directly translated after delivery to a cell without the need for an intervening replication step (e.g., reverse transcription). Translation of RNA results in the production of nonstructural proteins (NSPs) that combine to form a replicase complex (i.e., an RNA-dependent RNA polymerase). The replicase complex is a component of the sa-mRNA, which amplifies the original RNA to produce both antisense and sense transcripts, resulting in the production of multiple daughter RNAs and, subsequently, the encoded polypeptide of interest. For example, self-replicating RNAs include viral replicases (or viral polymerases).

[0119] For example, the sa-mRNA comprises an NSP derived from (or based on) an alphavirus. Exemplary alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEEV; e.g., Trinidad donkey, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, SAAR86 virus, Everglades virus, Mucambo virus, Barmah Forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. The term alphavirus also includes chimeric alphaviruses, which contain genomic sequences from more than one alphavirus (as described by Perri et al., (2003) J. Virol. 77(19):10394-403). In another example, the self-replicating RNA is derived from or based on a virus other than an alphavirus (e.g., a positive-strand RNA virus). Suitable positive-strand RNA viruses suitable for use in the present disclosure will be apparent to those of skill in the art and include, for example, a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus.

[0120] Exemplary sa-mRNAs also include a subgenomic (SG) promoter, which, upon binding to a nucleotide sequence encoding an NSP and / or a polypeptide of interest, drives expression of the NSP and / or polypeptide of interest. The present disclosure provides a self-replicating RNA comprising a nucleotide sequence encoding an antigen operably linked to an SG promoter. SG promoters (also known as "junction region" promoters) suitable for use in the present disclosure will be apparent to those skilled in the art and / or are described herein. In one example, the SG promoter is derived from or based on an alphavirus SG promoter. For example, the SG promoter is a native alphavirus SG promoter. In one embodiment, the native SG promoter is a minimal SG promoter. For example, a minimal SG promoter is the minimal sequence required for transcription initiation. In one example, the self-replicating RNA comprises nonstructural proteins, 5' and 3' untranslated regions (UTRs), and a native subgenomic promoter of an RNA virus. In another example, the self-replicating RNA comprises 5'- and 3'-terminal UTRs of an RNA virus. In one example, the mRNA is a self-replicating RNA, such as a monocistronic or bicistronic self-replicating RNA as described in PCT / IB2021 / 061203.

[0121] An mRNA useful for formulation with LNPs may include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5' end of the first region (e.g., the 5'-UTR), a second flanking region located at the 3' end of the first region (e.g., the 3'-UTR), at least one 5' cap region, and a 3' stabilization region. In some examples, the mRNA further includes a polyA tract and / or a Kozak sequence (e.g., within the 5'-UTR). In some cases, the mRNA may include one or more intron sequences that can be excised from the mRNA. In some examples, the mRNA may include a 5' cap structure, chain-terminating nucleotides, a stem-loop, a polyA sequence, and / or a polyadenylation signal. Any one of the regions of the mRNA may include one or more alternative components (e.g., alternative nucleosides). For example, the 3'-stabilization region may include alternative nucleosides, such as L-nucleosides, inverted thymidines, or 2'-O-methyl nucleosides, and / or the coding region, 5'-UTR, 3'-UTR, or the cap region may include alternative nucleosides, such as 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine), and / or 5-substituted cytidines (e.g., 5-methyl-cytidine).

[0122] In some examples, the mRNA may include one or more intron sequences that can be excised from the mRNA.

[0123] In some embodiments, the length of the mRNA is greater than 300 nt, e.g., greater than 500 nt or greater than 1000 nt. In one embodiment, the length of the mRNA is between 500 nt and 20,000 nt. In one embodiment, the length of the mRNA is between 500 nt and 10,000 nt. In one embodiment, the length of the mRNA is between 10,000 nt and 20,000 nt. In one embodiment, the length of the mRNA is between 5,000 nt and 20,000 nt. In one embodiment, the length of the mRNA is between 10,000 nt and 15,000 nt.

[0124] mRNA can be naturally occurring or non-naturally occurring. mRNA suitable for use in the LNPs of the invention can include one or more naturally occurring components, including any of the standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one example, all or substantially all of the nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3'-UTR, (d) the poly-A tail, and any combination of (a, b, c, or d above) include the naturally occurring standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).

[0125] In some examples, mRNAs may contain one or more alternative components that confer useful properties, including increased stability and / or lack of substantial induction of an innate immune response in cells into which the mRNA is introduced, as described herein. For example, alternative mRNAs exhibit reduced degradation in cells into which the mRNA is introduced, compared to the corresponding unmodified mRNA. These alternative species may increase the efficiency of protein production, intracellular retention of the mRNA, and / or viability of contacted cells, and may also be less immunogenic.

[0126] mRNA can include one or more modified (e.g., altered or alternative) nucleobases, nucleosides, nucleotides, or combinations thereof. mRNA can include any useful modification or alteration to the nucleobases, sugars, or internucleoside linkages (e.g., phosphate linkages / phosphodiester linkages / phosphodiester backbones), etc. In some examples, one or more modifications are present in each of the nucleobases, sugars, and internucleoside linkages.

[0127] An mRNA may be uniformly modified or unmodified along the entire length of the molecule, for example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or any one or more or all of A, G, U, C) may be uniformly modified or unmodified in the mRNA or in a given, predetermined sequence region thereof.

[0128] Different sugar modifications and / or internucleoside linkages (e.g., backbone structures) can be present at various positions within an mRNA. One skilled in the art will appreciate that nucleotide analogs or other modification(s) can be placed at any position(s) within the mRNA as long as mRNA function is not substantially impaired. Modifications can be at the 5' or 3' terminus. In some examples, the mRNA includes a modification at the 3' terminus.

[0129] Alternative nucleosides and nucleotides may include alternative nucleic acid bases. The nucleic acid bases of mRNA are organic bases such as purines or pyrimidines, or derivatives thereof. The nucleic acid bases may be standard bases (e.g., adenine, guanine, uracil, thymine, and cytosine). These nucleic acid bases may be modified or completely substituted to provide mRNA molecules with enhanced properties, for example, improved stability, such as nuclease resistance. Non-standard or modified bases may include, for example, one or more substitutions or modifications, including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction.

[0130] Alternative nucleotide base pairs include not only canonical adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and / or alternative nucleotides containing non-standard or alternative bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard and standard bases, or between two complementary non-standard base structures. One example of such a non-standard base pair is a base pair between the alternative nucleotide inosine and adenine, cytosine, or uracil.

[0131] In some embodiments, the nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having an alternative uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio-uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m3U). , 5-methoxy-uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm5s2U), 5-aminomethyl-2 -thio-uracil (nm5s2U), 5-methylaminomethyl-uracil (mnm5U), 5-methylaminomethyl-2-thio-uracil (mnm5s2U), 5-methylaminomethyl-2-seleno-uracil (mnm5se2U), 5-carbamoylmethyl-uracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uracil (cmnm5s2U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τ m5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (mψ), 1-ethyl-pseudouridine (Et1ψ), 5-methyl-2-thio-uracil (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy- Pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm5s2U), 5,2'-O 5-(isopentenylaminomethyl)-2'-O-methyluridine (cmnm5Um), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyluridine (mcm5Um). Examples of modified uracils include '-O-methyluridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyl-uracil, 5-methoxy-2-thiouracil, and 5-[3-(1-E-propenylamino)]uracil. In one example, the modified uracil is pseudouridine. In one example, the modified uracil is N1-methyl-pseudouridine.

[0132] In some embodiments, the nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides having alternative cytosines include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine). , 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza- Pseudoisocytidine, Zebularine, 5-Aza-Zebularine, 5-Methyl-Zebularine, 5-Aza-2-Thio-Zebularine, 2-Thio-Zebularine, 2-Methoxy-Cytosine, 2-Methoxy-5-Methyl-Cytosine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, Lysidine (k2C), 5,2'-O-Dimethyl-Cytidine (m5Cm), N4- These include acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine. In one example, the modified cytosine is 5-methyl-cytosine.

[0133] In some embodiments, the nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza cis-2,6-diaminopurine, 1-methyl-adenine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarba moyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl These include adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0134] In some embodiments, the nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (mII), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o2yW), hydroxywyosine (OHyW), undermodified hydroxywyosine (OHyW*), 7-deaza-guanine, queuosine ( Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQ0), 7-aminomethyl-7-deaza-guanine (preQ1), archeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7 -methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2- These include dimethyl-6-thioguanine, N2-methyl-2'-O-methylguanosine (m2Gm), N2,N2-dimethyl-2'-O-methylguanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (mlm), 1-thio-guanine, and O-6-methyl-guanine.

[0135] The alternative nucleobase of a nucleotide can independently be a purine, a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can be substituted for adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, nucleobases also include, for example, derivatives of natural and synthetic bases, such as pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl , 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; or 1,3,5 triazine.

[0136] Enriched populations of LNPs prepared using the methods described herein can, in some examples, be used to induce expression of a desired protein, both in vitro and in vivo, by contacting cells with the enriched population of LNPs, where the LNPs encapsulate mRNA that is expressed to produce the desired protein, such as mRNA encoding the desired protein.

[0137] The mRNA of the present disclosure typically comprises a nucleotide sequence encoding a polypeptide of interest. The nucleotide sequence can encode any polypeptide known to those of skill in the art, including any naturally occurring, non-naturally occurring, or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some examples, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell. In one example, the nucleotide sequence encodes an antigen (e.g., a pathogenic antigen). For example, the antigen can induce an immune response in a subject. In one example, the mRNA of the present disclosure comprises a nucleotide sequence encoding an antigen derived from a virus. In one example, the mRNA of the present disclosure comprises a nucleotide sequence encoding an antigen derived from a respiratory virus, e.g., influenza virus, coronavirus, or respiratory syncytial virus (RSV). In one example, the mRNA comprises a nucleotide sequence encoding an antigen described herein.

[0138] mRNA for formulation with LNPs can be prepared according to any available technique known in the art. For example, mRNA can be prepared by enzymatic synthesis, which provides a template-directed synthesis process for RNA molecules from an engineered DNA template composed of an upstream bacteriophage promoter sequence linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from many sources using suitable techniques known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD, In RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012).

[0139] RNA transcription is carried out in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing the possibility of degradation of the resulting mRNA transcript. In vitro transcription can be carried out using various commercially available kits, including but not limited to, RiboMax Large Scale RNA Production System (Promega) and MegaScript Transcription Kit (Life Technologies), as well as commercially available reagents such as RNA polymerase and rNTPs. Methodologies for in vitro transcription of mRNA are well known in the art. (See, e.g., Losick, R., 1972, In Vitro Transcription, Ann Rev Biochem v. 41 409-46; Kamaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by in vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114, all of which are incorporated herein by reference.) After in vitro transcription, the DNA template can be removed, for example, by DNase digestion. Those skilled in the art will understand that synthetic mRNA capping is performed to correct mRNA processing and contribute to mRNA stabilization.In one embodiment, the mRNA is enzymatically 5'-capped. In one embodiment, the mRNA is co-transcriptionally capped. For example, the 5' cap is a cap0 structure or a cap1 structure. In one embodiment, the 5' cap is a cap0 structure, e.g., a 5'-cap (i.e., cap0) consists of an inverted 7-methylguanosine connected to the rest of the mRNA via a 5'-5' triphosphate bridge. In one embodiment, the 5' cap is a cap1 structure, e.g., a 5'-cap (i.e., cap1) consists of a cap0 with an additional methylation at the 2'0 position of the initiating nucleotide.

[0140] In one embodiment, the desired in vitro transcribed mRNA is then purified from undesired components of transcription or related reactions. Techniques for isolating mRNA transcripts are well known in the art, including phenol / chloroform extraction, precipitation with either alcohol or lithium chloride in the presence of monovalent cations, or chromatography. In another embodiment, mRNA is purified using tangential flow filtration (TFF). After purification, the mRNA is resuspended, for example, in nuclease-free water.

[0141] Encapsulation Rate Methods for producing LNPs used by those skilled in the art can produce mixed or heterogeneous LNP populations. Mixed or heterogeneous LNP populations can include RNA partially encapsulated within the LNPs, RNA associated with the surface of the LNPs, and / or RNA not associated with the LNPs. An "enriched" LNP population or preparation refers to an LNP population derived from a starting LNP population (e.g., a heterogeneous LNP population, such as one prepared by nanoprecipitation) that contains a higher proportion of LNP-encapsulated RNA than the proportion of LNP-encapsulated RNA in the starting population. For example, the starting LNP population can be enriched for LNPs containing fully encapsulated RNA. As used herein, the terms "LNP population" and "LNP preparation" are used interchangeably.

[0142] As used herein, the "encapsulation rate" of a population refers to the amount of RNA that is completely encapsulated within the LNP relative to the total amount of RNA present in the LNP population. As used herein, "completely encapsulated" refers to complete enclosure, confinement, surrounding, or envelopment. For example, if 92 mg of RNA present in an LNP population is completely encapsulated within the LNP out of a total of 100 mg of RNA present in the population, the encapsulation efficiency can be expressed as 92%.

[0143] The encapsulation rate of the LNP population prior to use in the methods of the disclosure can be at least 10%, e.g., about 10%, 15%, 20%, 25%, 20%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the encapsulation rate prior to use in the methods described herein can be at least 10%. In certain embodiments, the encapsulation rate can be at least 20%. The encapsulation rate of the LNP population prior to use in the methods of the present disclosure can be less than 10%, e.g., less than about 10%, 15%, 20%, 25%, 20%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the encapsulation rate prior to use in the methods described herein can be less than 10%. In certain embodiments, the encapsulation rate can be less than 20%. In certain embodiments, the encapsulation rate can be less than 50%.

[0144] As will be appreciated, the encapsulation rate of an enriched population of LNPs will be higher than the encapsulation rate of a non-enriched population (i.e., a population of LNPs prior to use in the methods of the present disclosure). The encapsulation rate of an enriched population of LNPs (e.g., produced by the methods of the present disclosure) can be at least 10%, e.g., about 10%, 15%, 20%, 25%, 20%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the encapsulation rate can be at least 80%. In certain embodiments, the encapsulation rate can be at least 90%. In certain embodiments, the encapsulation rate can be at least 95%.

[0145] The amount of RNA present in an LNP population can be measured using techniques known to those skilled in the art. For example, the amount of RNA contained in an LNP population can be measured using a fluorescent assay using a dye (e.g., Ribogreen) that increases in emission intensity upon binding to RNA, or by measuring absorbance at 260 nm. Using the Ribogreen assay as an example, the total amount of RNA is determined by disrupting the LNPs with detergent to expose the encapsulated RNA, adding the dye, and comparing the emission intensity to a standard curve generated using ribosomal RNA. Previously, it was thought that if detergent disruption of the LNPs were omitted from the assay, the amount of unencapsulated RNA could be estimated in a similar manner. However, the inventors have discovered that this underestimates the amount of unencapsulated mRNA, and that an alternative assay is needed to estimate encapsulated mRNA in an LNP formulation or population.

[0146] In one example, the encapsulation rate can be determined by comparing the total amount of RNA in the composition before and after contact with the anion exchanger. In one example, the encapsulation rate can be determined using the following formula: Encapsulation rate (%) = (RNA 未結合 ) / RNA ロード) x 100 In the formula, RNA ロード and RNA 未結合 are the absolute amount of RNA in the unbound fraction and the absolute amount of RNA loaded onto the anion exchange column, respectively.

[0147] Encapsulation Efficiency As used herein, "encapsulation efficiency" refers to the amount of mRNA that becomes part of an LNP composition relative to the initial total amount of mRNA used to prepare the LNP composition. For example, if an LNP formulation contains 92 mg of mRNA and 100 mg of mRNA is initially provided to form the composition, the encapsulation efficiency may be 92%. This differs from the encapsulation rate, which refers to the amount of mRNA that is completely encapsulated within the LNPs in the formulation relative to the total amount of mRNA present in the formulation.

[0148] Lipid Nanoparticle Composition The present disclosure provides compositions comprising an enriched population of LNPs prepared by the methods described herein.

[0149] The present disclosure also provides a pharmaceutical composition comprising an enriched population of lipid nanoparticles prepared by the methods described herein and a pharmaceutically acceptable carrier.

[0150] Compositions containing enriched populations of lipid nanoparticles prepared by the methods described herein can be formulated for administration via any acceptable method for administering lipid particles. The pharmaceutical compositions described herein can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical LNP compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques. In one embodiment, LNPs are administered parenterally, such as intramuscularly, subcutaneously, or intravenously. In some embodiments, LNPs are administered intramuscularly.

[0151] The composition that is administered to the subject can be in the form of one or more dosage forms, for example, the volume of tablet or injectable liquid can be a single dosage form.The actual method of preparing such dosage forms is known or will be clear to those skilled in the art.See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

[0152] Thus, in one embodiment, a composition (e.g., a pharmaceutical composition) is provided that includes an enriched population of lipid nanoparticles in combination with a pharmaceutically acceptable carrier. The composition may optionally include a pharmaceutically acceptable excipient. Terms such as "pharmaceutically acceptable carrier, diluent, or excipient" refer to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving an active compound) that has substantially non-toxic and non-inflammatory properties in a patient.

[0153] Generally, a "carrier" refers to a solid or liquid filler, binder, diluent, encapsulating material, emulsifier, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely administered to any subject, for example, a human. Depending on the particular route of administration, a variety of acceptable carriers known in the art can be used, for example, as described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0154] Excipients may include, for example: anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (glidants), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other species disclosed herein.

[0155] The formulation of the LNP to be administered will vary depending on the selected route of administration and formulation (e.g., solution, emulsion, capsule). A suitable pharmaceutical composition containing the LNP to be administered can be prepared in a pharmaceutically acceptable carrier. In the case of a solution or emulsion, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose solution, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. A variety of suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles can include various additives, preservatives, fluid, nutrient, or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions of the invention can optionally contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions (pH adjusting agents, buffering agents, and toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.) The LNPs can be stored in a liquid state or can be lyophilized for storage and reconstituted with a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.

[0156] When the LNP composition is a vaccine composition, the carrier can be water, typically pyrogen-free water; isotonic saline; or a buffered (aqueous) solution, such as a phosphate, citrate, or other buffer solution. For injection of the LNP vaccine composition, water, or preferably a buffer, more preferably an aqueous buffer, containing sodium salts, preferably at least 50 mM sodium salts, calcium salts, preferably at least 0.01 mM calcium salts, and optionally potassium salts, e.g., at least 3 mM potassium salts, may be used. In one example, the sodium salts, calcium salts, and optionally potassium salts may be present as their chlorides, iodides, or bromides, or in the form of their hydroxides, carbonates, bicarbonates, or sulfates, etc. Non-limiting examples of sodium salts include, for example, NaCl, NaI, NaBr, Na2CO3, NaHCO3, and Na2SO4; examples of any potassium salt include, for example, KCl, KI, KBr, K2CO3, KHCO3, and K2SO4; and examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO3, CaSO4, and Ca(OH)2. Additionally, organic anions of the above cations may be included in the buffer. In certain embodiments, a buffer suitable for injection purposes may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2), and optionally potassium chloride (KCl), and additional anions in addition to chloride may be present. In embodiments, the salts in the injection buffer are present in concentrations of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl), and at least 0.01 mM calcium chloride (CaCl2). The injection buffer may be hypertonic, isotonic, or hypotonic relative to a particular reference medium.

[0157] In one example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain examples, a pharmaceutical composition of the present disclosure has a pH of about 7-8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or 7.5-8 or 7-7.8). For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5-8, which is suitable for storage and / or transportation, e.g., at about -20°C. For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and PBS, has a pH of about 7-7.8, and is suitable for storage and / or transport, for example, at about 4° C. or below. "Stability," "stabilized," and "stable," in the context of the present disclosure, refer to the resistance of the nanoparticle composition and / or pharmaceutical composition disclosed herein to chemical or physical changes (e.g., degradation, change in particle size, aggregation, change in encapsulation, etc.) when subjected to stresses, such as shear forces, freeze / thaw stresses, etc., under given conditions of manufacture, preparation, transport, storage, and / or use.

[0158] When the LNP composition is a vaccine composition, it may further comprise one or more pharmaceutically acceptable adjuvants to enhance the immunostimulatory properties of the composition. The adjuvant may be any compound suitable for aiding in the administration and delivery of the LNP composition and capable of initiating or augmenting an immune response of the innate immune system, i.e., a non-specific immune response. Such adjuvants are known to those skilled in the art and may be selected from any adjuvant suitable for the particular properties of the vaccine, i.e., the induction of an appropriate immune response in a mammal.

[0159] In one example, the pharmaceutical compositions described herein may comprise a total lipid content of about 0.1 mg to 10 mg, or 0.5 mg to 8 mg, or 0.7 mg to 6 mg, or 0.7 mg to 2 mg. In some examples, such immunogenic compositions may comprise a total lipid content of about 1 mg / mL to 15 mg / mL, or 2 mg / mL to 10 mg / mL, or 2.5 to 5 mg / mL.

[0160] The pharmaceutical compositions described herein may be provided as a frozen concentrate for injectable solution. In one example, to prepare the injectable solution, the frozen concentrate is thawed and diluted with an isotonic solution (e.g., 0.9% NaCl, saline), for example, by a one-step dilution process. In some examples, bacteriostatic sodium chloride solution (e.g., 0.9% NaCl, saline) cannot be used as a diluent. In some examples, the diluted composition is an off-white suspension. The concentration of the final injectable solution will vary depending on the respective dose level administered.

[0161] The compositions described herein can be transported and / or stored under temperature-controlled conditions, e.g., at temperatures below about 4-5°C, below about -20°C, or at temperatures below -70°C ± 10°C (e.g., -80°C to -60°C), using, for example, a cooling system (which may be or include, e.g., dry ice) to maintain the desired temperature. In one example, the compositions described herein are transported in temperature-controlled thermal shippers. Such shippers may contain GPS-enabled thermal sensors to track the location and temperature of each package. The compositions can be stored, for example, by replenishing with dry ice.

[0162] Dosage When formulated, the compositions of the present disclosure are administered in a manner compatible with the dosage formulation and in an amount that is therapeutically / prophylactically effective. The dosage range for administering the enriched population of LNPs or compositions thereof of the present disclosure is large enough to produce the desired effect. For example, the composition comprises an effective amount of mRNA. In one example, the composition comprises a therapeutically effective amount of mRNA. In another example, the composition comprises a prophylactically effective amount of mRNA.

[0163] The dosage should not be so large as to cause side effects. Generally, the dosage will vary depending on the age, condition, sex, and degree of disease in the patient, but can be determined by those skilled in the art. The dosage can be adjusted by an individual physician if any complications arise.

[0164] Dosages can vary from about 0.1 mg / kg to about 300 mg / kg, for example, about 0.2 mg / kg to about 200 mg / kg, for example, about 0.5 mg / kg to about 20 mg / kg, administered once or more times a day for one to several days.

[0165] In some instances, the mRNA is administered at an initial (or loading) dose, which is higher than subsequent (maintenance) doses. For example, the mRNA is administered at an initial dose of about 10 mg / kg to about 30 mg / kg. The mRNA is then administered at maintenance doses of about 0.0001 mg / kg to about 10 mg / kg. The maintenance doses can be administered every 7 to 35 days (e.g., every 7, 14, or 28 days).

[0166] In some embodiments, a dose escalation regimen is used, in which the mRNA is initially administered at a lower dose than is used in subsequent doses. This dosing regimen is useful if the subject is initially experiencing an adverse event.

[0167] If the subject does not respond adequately to treatment, multiple doses per week may be administered. Alternatively, or additionally, increased doses may be administered.

[0168] A subject can be re-treated with an enriched LNP population of the present disclosure. The subject can be re-treated with the enriched LNP population by administering multiple exposures or dose sets, e.g., at least about 2 exposures of the LNP population, e.g., about 2-60 exposures, more particularly about 2-40 exposures, and most particularly about 2-20 exposures.

[0169] In one embodiment, optional re-treatment can occur upon recurrence of signs or symptoms of the disease. In another embodiment, optional re-treatment can occur at defined intervals. For example, subsequent exposures can occur at various intervals, such as about 24-28 weeks, or 48-56 weeks, or longer. For example, each exposure can occur at intervals of about 12-14 weeks, 24-26 weeks, about 38-42 weeks, or about 50-54 weeks.

[0170] For subjects who do not respond adequately to treatment, multiple doses may be administered weekly. Alternatively, or additionally, increased doses may be administered.

[0171] In another example, for subjects experiencing adverse reactions, the initial (or loading) dose can be administered on multiple days or on multiple consecutive days during the week.

[0172] In one example, the pharmaceutical composition or vaccine described herein may be administered as part of a regimen. In one example, the regimen administered to a subject may include or consist of a single dose. In one example, the regimen administered to a subject may include multiple doses (e.g., at least two doses, at least three doses, or more). In one example, the regimen administered to a subject may include a first dose and a second dose. In one example, the regimen consists of the administration of two doses of the composition. In one example, the first dose and the second dose are administered at least two weeks, at least three weeks, at least four weeks, or more apart. In one example, the doses may be at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, or more apart. In one example, the doses may be administered 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days apart. In one example, doses may be administered about 1 to about 3 weeks apart, or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, or about 1 to about 6 weeks apart, or about 1 to more than 6 weeks apart. In one example, doses may be spaced apart by a period of about 7 to about 60 days, such as about 14 to about 48 days. In one example, the minimum number of days between doses may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days, or more.In one example, the maximum number of days between doses may be about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 days or less. In one example, doses may be spaced apart by about 21 days to about 28 days. In one example, doses may be spaced apart by about 21 days to about 56 days. In one example, a first dose is a different amount than one or more subsequent doses. In one example, a composition described herein is administered (e.g., by intramuscular injection) as two doses 21 days apart. In one example, the compositions described herein are administered (eg, by intramuscular injection) as two doses 56 days apart.

[0173] Each dose may contain an amount of RNA that provides a therapeutically effective amount. In one example, a dose may contain sufficient RNA to elicit an immune response in a subject receiving at least one dose of the composition. In one example, a dose may contain 0.0001 μg to 300 μg, 0.001 μg to 200 μg, or 0.001 μg to 100 μg, e.g., about 0.001 μg, about 0.01 μg, about 0.1 μg, about 1 μg, about 3 μg, about 10 μg, about 30 μg, about 50 μg, or about 100 μg of RNA. In one example, a dose may contain 100 μg or less, 90 μg or less, 80 μg or less, 70 μg or less, 60 μg or less, 50 μg or less, 40 μg or less, 30 μg or less, 20 μg or less, 10 μg or less, 5 μg or less, 2.5 μg or less, or 1 μg or less of RNA. In one example, a dose may comprise at least 0.001 μg, at least 0.01 μg, at least 0.1 μg, at least 0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 10 μg, at least 20 μg, at least 30 μg, or at least 40 μg of RNA. In one example, an effective amount is about 100 μg of RNA per dose. In one example, an effective amount is about 30 μg of RNA per dose. In one example, an effective amount is about 10 μg of RNA per dose. In one example, an effective amount is about 5 μg of RNA per dose. In one example, an effective amount is about 3 μg of RNA per dose. In one example, an effective amount is about 1 μg of RNA per dose. In one example, at least two such doses are administered.

[0174] In one example, the mRNA is administered to the subject at a dose of 100 μg or less, 90 μg or less, 80 μg or less, 70 μg or less, 60 μg or less, 50 μg or less, 40 μg or less, 30 μg or less, 20 μg or less, 10 μg or less, or 5 μg or less. In one example, the mRNA is administered to the subject at a dose of 10 μg or less.

[0175] Without wishing to be bound by any particular theory, the present disclosure suggests that enriched LNP populations produced using the methods described herein may be particularly useful and / or effective for use as or in immunogenic compositions (e.g., vaccines) and / or for achieving the immunological effects described herein (e.g., generation of neutralizing antibodies and / or T cell responses (e.g., CD4+ and / or CD8+ T cell responses)).

[0176] In one example, the amount (i.e., dose) of mRNA administered is effective to induce an immune response in the subject, and the amount of RNA administered is sufficient to induce an immune response in the subject at a dose at least 2-fold (e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold) lower than a reference composition that has not been treated with an anion exchanger. In one example, the subject is a mouse model.

[0177] In one example, the dose comprises less than 100 μg (e.g., less than 50 μg, less than 40 μg, or less than 30 μg) of mRNA, and the composition elicits an immune response greater than the immune response elicited by a reference composition comprising at least 100 μg of mRNA that has not been treated with an anion exchanger.

[0178] In one example, the immune response can include the generation of binding antibody titers to one or more antigens encoded by the mRNA (e.g., a coronavirus protein or fragment thereof, or an influenza protein or fragment thereof). In one example, the immune response can include the generation of binding antibody titers to the coronavirus spike (S) protein and / or nucleocapsid (N) protein (e.g., a SARS-CoV-2 N protein and / or S protein). In one example, the immune response can include the generation of binding antibody titers to the SARS-CoV-2 N protein and / or S protein from SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020. In one example, the immune response can include the generation of binding antibody titers to influenza A virus strain proteins (e.g., influenza A virus hemagglutinin (HA) protein, neuraminidase (NA) protein, matrix (M) protein, nucleoprotein (NP), nonstructural (NS) protein, or immunogenic fragments or variants thereof). In one example, the immune response can include the production of binding antibody titers against the H5 hemagglutinin protein, the M1 matrix protein, and / or the N1 neuraminidase protein.

[0179] In one example, the immune response can include the generation of neutralizing antibody titers against one or more antigens encoded by the mRNA (e.g., a coronavirus protein or fragment thereof, or an influenza protein or fragment thereof). In one example, the immune response can include the generation of neutralizing antibody titers against the coronavirus spike (S) protein and / or nucleocapsid (N) protein (e.g., the SARS-CoV-2 N protein and / or S protein). In one example, the immune response can include the generation of neutralizing antibody titers against the SARS-CoV-2 N protein and / or S protein from SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020. In one example, the immune response can include the generation of neutralizing antibody titers against influenza A virus strain proteins (e.g., influenza A virus hemagglutinin (HA) protein, neuraminidase (NA) protein, matrix (M) protein, nucleoprotein (NP), nonstructural (NS) protein, or immunogenic fragments or variants thereof). In one example, the immune response can include the generation of neutralizing antibody titers against the H5 hemagglutinin protein, the M1 matrix protein, and / or the N1 neuraminidase protein. In one example, the compositions described herein are demonstrated to achieve neutralizing antibody titers in an appropriate system (e.g., humans infected with SARS-CoV-2 / influenza and / or populations thereof, and / or model systems thereof). For example, in some examples, such neutralizing antibody titers may be demonstrated in one or more of a human population, a non-human primate model (e.g., rhesus macaque), and / or a mouse model. In one example, such neutralizing antibody titers may be demonstrated in a mouse model.

[0180] In one example, the neutralizing antibody titer is sufficient (e.g., demonstrated to be sufficient) to reduce viral infection of B cells compared to that observed in an appropriate control (e.g., an unvaccinated control subject, or a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In one such example, such reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[0181] In one example, the neutralizing antibody titer is sufficient (e.g., demonstrated to be sufficient) to reduce the rate of asymptomatic viral infection compared to that observed in an appropriate control (e.g., unvaccinated control subjects or subjects vaccinated with a live-attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In one such example, such reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In one example, such reduction can be characterized by evaluation of protein serology, e.g., SARS-CoV-2 N protein serology.

[0182] In one example, the neutralizing antibody titer is sufficient (e.g., demonstrated to be sufficient) to reduce or block viral fusion with epithelial cells and / or B cells in a vaccinated subject compared to that observed in an appropriate control (e.g., an unvaccinated control subject, or a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In one such example, such reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[0183] In one example, the induction of neutralizing antibody titers may be characterized by an increase in B cell numbers, which in some examples may include plasma cells, class-switched IgG1- and IgG2-positive B cells, and / or germinal center B cells. In some examples, the provided immunogenic compositions have been demonstrated to achieve such an increase in B cell numbers in an appropriate system (e.g., humans infected with SARS-CoV-2 / influenza and / or populations thereof, and / or model systems thereof). For example, such an increase in B cell numbers may be demonstrated in one or more human populations, non-human primate models (e.g., rhesus macaques), and / or mouse models. In one example, such an increase in B cell numbers may be demonstrated in the draining lymph nodes and / or spleen of a mouse model (e.g., at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days) following immunization of such mouse model with a provided immunogenic composition.

[0184] In one example, the induction of neutralizing antibody titers can be characterized by a reduction in the number of circulating B cells in the blood. In one example, the provided immunogenic compositions have been demonstrated to achieve such a reduction in the number of circulating B cells in the blood in an appropriate system (e.g., humans infected with SARS-CoV-2 / influenza and / or populations thereof, and / or model systems thereof). For example, such a reduction in the number of circulating B cells in the blood may be demonstrated in one or more human populations, non-human primate models (e.g., rhesus macaques), and / or mouse models. In one example, such a reduction in the number of circulating B cells in the blood may be demonstrated in a mouse model (e.g., at least 7 days, at least 14 days, at least 21 days, at least 28 days, at least 35 days, at least 42 days, or at least 49 days) after immunization of such mouse model with a composition described herein.

[0185] In one example, a regimen described herein (e.g., one or more doses of a composition described herein) can induce an antibody response within 21 days after vaccination. In one example, such an antibody response can include a total IgG level of 100-20,000 (e.g., 300-10,000) as assessed by ELISA in an animal model (e.g., a mouse model) measured 21 days after vaccination with a 0.001-1 μg dose. In one example, a regimen described herein (e.g., one or more doses of a composition described herein) can induce a pseudotype virus neutralization titer of 15,000-55,000 as measured in an animal model (e.g., a mouse model) 42 days after vaccination with a 0.001-1 μg dose. In one example, a regimen described herein (e.g., one or more doses of a composition described herein) may induce a hemagglutination inhibition titer (HAI) of greater than 1:40, or greater than 1:80, as measured in an animal model (e.g., a mouse model, e.g., a BALB / c mouse). In one example, a regimen described herein (e.g., one or more doses of a composition described herein) may induce a hemagglutination inhibition titer (HAI) of greater than 500, greater than 600, greater than 700, greater than 800, or greater than 900, as measured in an animal model (e.g., a mouse model), 42 days post-vaccination.

[0186] In one example, a regimen described herein (e.g., one or more doses of a composition described herein) can expand an antigen-specific CD8 and / or CD4 T cell response by at least 50% or more (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more) compared to that observed in the absence of such a composition. In one example, a regimen described herein (e.g., one or more doses of a composition described herein) can expand an antigen-specific CD8 and / or CD4 T cell response by at least 1.5-fold or more (e.g., at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more) compared to that observed in the absence of such a composition.

[0187] In one example, a regimen described herein (e.g., one or more doses of a composition described herein) can expand T cells exhibiting a Th1 phenotype (e.g., as characterized by expression of IFN-γ, IL-2, IL-4, and / or IL-5) by at least 50% or more (including, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more) compared to that observed in the absence of such compositions. In one example, a regimen described herein (e.g., one or more doses of a composition described herein) can expand T cells exhibiting a Th1 phenotype (e.g., characterized by expression of IFN-γ, IL-2, IL-4, and / or IL-5) by, for example, at least 1.5-fold or more (e.g., at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more). In some examples, the T cell phenotype may be or include a predominantly Th1 cytokine profile (e.g., characterized as being INF-γ positive and / or IL-2 positive) and / or may have no or biologically insignificant IL-4 secretion.

[0188] In one example, a regimen described herein (e.g., one or more doses of a composition described herein) induces and / or achieves the production of antigen-specific CD4+ T cells. In one example, characterization of CD4+ and / or CD8+ T cell responses (e.g., as described herein) in a subject administered a composition described herein can be performed using an ex vivo assay using PBMCs collected from the subject. In one example, the immunogenicity of the mRNA compositions described herein can be assessed by one or more of the following serological immunogenicity assays: detection of IgG, IgM, and / or IgA against the protein encoded by the mRNA present in a blood sample from a subject administered a provided mRNA composition, and / or a neutralization assay using appropriate pseudotyped and / or wild-type viruses.

[0189] In one example, a composition described herein (e.g., when administered to a relevant population) may provide an improved therapeutic outcome (e.g., an effective immune response and / or detectable expression of an encoded protein or immunogenic fragment thereof) at one or more doses compared to a composition that has not been treated with an anion exchanger prior to administration. In one example, certain results may be achieved at lower doses (e.g., a dose of 0.001 μg in a mouse model) than those required for a composition that has not been treated with an anion exchanger prior to administration.

[0190] In one example, the compositions and / or methods described herein can provide an antigen neutralization geometric mean titer that is at least 1.5-fold or greater (including, for example, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, or greater) compared to the neutralization GMT of a control composition not treated with an anion exchanger, measured 42 days after the first dose or 21 days after the second dose. In one example, an increase in antigen neutralization geometric mean titer can be achieved at low doses (e.g., a dose of 0.001 μg in a mouse model).

[0191] In one example, the compositions and / or methods described herein can provide an in vitro potency that is at least 1.5-fold or greater (including, for example, at least 2-fold, at least 3-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 8-fold, at least 10-fold, or more) compared to the in vitro potency of a comparable composition that has not been treated with an anion exchanger. In one example, the increase in in vitro potency can be achieved at low doses (e.g., a dose of 0.001 μg in a mouse model). In vitro potency can be measured using any method known to one of skill in the art. In one example, in vitro potency is measured as described herein.

[0192] In one example, the titer of binding and / or neutralizing antibodies produced in mice vaccinated with at least one dose of a composition described herein is increased by at least 1 log compared to a control, where the control is the titer of binding and / or neutralizing antibodies produced in mice administered a composition that has not been contacted with an anion exchanger. In one example, an increase in the titer of binding and / or neutralizing antibodies can be achieved at low doses (e.g., a dose of 0.001 μg).

[0193] In one example, the titer of binding and / or neutralizing antibodies produced in mice vaccinated with at least one dose of a composition described herein is increased by at least two-fold compared to a control, where the control is the titer of binding and / or neutralizing antibodies produced in mice administered a composition that has not been contacted with an anion exchanger. In one example, the increase in titer of binding and / or neutralizing antibodies can be achieved at low doses (e.g., a dose of 0.001 μg).

[0194] Treatment or prevention methods Compositions comprising enriched populations of LNPs described herein can be used to treat and / or prevent diseases, disorders, and / or conditions, which may include, but are not limited to, rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0195] The LNP compositions can be formulated in unit dosage form. Additionally, the therapeutically or prophylactically effective amount for any particular patient will depend on a variety of factors, including the severity and identity of the disorder being treated; the particular composition utilized; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the particular pharmaceutical composition utilized; the duration of treatment; the particular pharmaceutical composition utilized, the drugs used in combination or co-administration; and similar factors well known in the medical arts.

[0196] The LNP compositions described herein may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents, which may be administered together in a single composition or separately in different compositions.

[0197] The enriched population of LNPs described herein can be used in a method for producing a polypeptide of interest in mammalian cells. The method for producing a polypeptide includes contacting cells with an enriched population of LNPs described herein, which contains mRNA encoding the polypeptide of interest. When cells are contacted with an enriched population of LNPs, the mRNA can be taken up into the cell and translated to produce the polypeptide of interest.

[0198] The step of contacting the enriched population of LNPs with cells may involve or result in transfection. Phospholipids contained in the lipid component of the LNP composition may facilitate transfection and / or increase transfection efficiency, for example, by interacting with and / or fusing with cell membranes or intracellular membranes. Transfection may result in translation of mRNA within the cell.

[0199] In some examples, the LNP compositions described herein can be used therapeutically. For example, the mRNA included in the LNP composition can encode (e.g., in a translatable region) a therapeutic polypeptide and produce the therapeutic polypeptide upon contact and / or entry into (e.g., transfection with) a cell. In other examples, the mRNA included in the LNP composition can encode a polypeptide that can improve or increase a subject's immunity.

[0200] In some embodiments, the mRNA contained in the LNP composition may encode a recombinant polypeptide that can replace one or more polypeptides that may be substantially absent in cells contacted with the LNP composition. The one or more substantially absent polypeptides may be absent due to a genetic mutation in the encoding gene or its regulatory pathway. Alternatively, the recombinant polypeptide produced by translation of the mRNA may antagonize the activity of an endogenous protein present within the cell, on the cell surface, or secreted from the cell. Antagonistic recombinant polypeptides may be desirable to counteract deleterious effects caused by the activity of endogenous proteins, such as altered activity or localization caused by mutations. In another alternative, the recombinant polypeptide produced by translation of the mRNA may indirectly or directly antagonize the activity of a biological moiety present within the cell, on the cell surface, or secreted from the cell. Antagonistic biological moieties include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins. The recombinant polypeptide produced by translation of the mRNA may be engineered to localize within a specific compartment within the cell, such as the nucleus, or may be engineered to be secreted from the cell or translocated to the plasma membrane of the cell.

[0201] In some examples, contacting cells with an LNP composition containing mRNA can reduce the cellular innate immune response to an exogenous polynucleotide. Cells may be contacted with a first LNP composition containing a first amount of a first exogenous mRNA containing a translatable region, and the level of the cellular innate immune response to the first exogenous mRNA may be determined. Subsequently, the cells may be contacted with a second LNP composition containing a second amount of the first exogenous mRNA, the second amount being a lower amount of the first exogenous mRNA compared to the first amount. Alternatively, the second composition may contain a first amount of a second exogenous mRNA that is different from the first exogenous mRNA. The step of contacting cells with the first and second LNP compositions may be repeated one or more times. Furthermore, the efficiency of polypeptide production (e.g., translation) in the cells may optionally be determined, and the cells may be repeatedly re-contacted with the first and / or second compositions until a target protein production efficiency is achieved.

[0202] In some examples, the present disclosure provides use of a composition comprising an enriched population of LNPs produced by a method described herein in the manufacture of a medicament for treating a disease, disorder, or condition, which may be as described in any one or more examples herein.

[0203] The medicament may be for the prevention or treatment of cancer, infectious diseases, allergies, or autoimmune diseases. In an embodiment, the medicament is a vaccine. The vaccine may be a tumor vaccine, an influenza vaccine, or a SARS-CoV-2 vaccine.

[0204] The present disclosure also provides a method of treating, preventing, or delaying the progression of a disease or condition in a subject, comprising administering an enriched population of LNPs or a composition comprising the enriched population of LNPs, for example, the disease or condition is selected from the group consisting of SARS-CoV-2 infection, COVID-19, ARDS, and combinations thereof.

[0205] In one embodiment, a method of generating an immune response in a subject is provided, the method comprising administering to the subject an enriched population of LNPs in an amount of less than 10 μg of RNA, wherein the LNPs comprise an ionizable lipid, a phospholipid, a PEG-lipid, and a structural lipid, and at least 50% of the LNPs comprise RNA encapsulated within the LNPs. In one embodiment, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the LNPs comprise RNA encapsulated within the LNPs.

[0206] Use of LNPs in vaccines The enriched population of LNPs produced using the methods described herein can be a component of a vaccine. In one embodiment, the present disclosure provides a method of using the pharmaceutical composition of the present disclosure as a vaccine. Vaccines include compounds and preparations capable of providing immunity against one or more conditions associated with infectious diseases and may include mRNA encoding antigens and / or epitopes from infectious diseases. Vaccines also include compounds and preparations that induce an immune response against cancer cells and may include mRNA encoding antigens, epitopes, and / or neoepitopes from tumor cells. Compounds that induce an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species.

[0207] In examples, the mRNA encodes an antigenic peptide or protein, or a fragment, variant, or derivative thereof. The antigenic peptide or protein may be a pathogenic antigen, a tumor antigen, an allergic antigen, or an autoimmune autoantigen. Such a pathogenic antigen may be an antigen derived from a pathogenic organism, particularly a bacterial, viral, or protozoal (multicellular) pathogenic organism, that provokes an immunological response in a mammalian subject, such as a human. The pathogenic antigen may be a surface antigen, such as a protein or a fragment thereof, located on the surface of a virus, bacterium, or protozoan.

[0208] Pathogenic antigens of interest may include those derived from one or more of the following: Acinetobacter baumannii, Anaplasma spp., Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Area nobacteri urn haemolyticum, Ascaris lumbricoides, Aspergillus spp., Astroviridae, Babesia spp., Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocysts hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia spp., Brucella spp., Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter spp., Candida albicans, Candida spp., Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, QD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium spp., Clostridium tetani, Coccidioides spp., coronavirus, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium spp., cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4), Dientamoebafragilis, Ebola virus (EBOV), Echinococcus spp., Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia spp., Entamoeba histolytica, Enterococcus spp., Enterovirus spp., enteroviruses, mainly Coxsackie A virus and enterovirus 71 (EV71), Epidermophyton spp., Epstein-Barr virus (EBV), Escherichia coli O157:H7, O111 and O104:H4, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, flavivirus, Francisella tularensis, Fusobacterium spp., Geotrichum candidum, Giardia intestinalis, Gnathostoma spp., GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipaviruses (Hendra virus, Nipah virus), Hepatitis A virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania spp., Leptospira spp., Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malasseziaspp, Marburg virus, measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae (influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurella spp, Plasmodium spp, Pneumocystis jirovecii, poliovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia spp., Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, rotavirus, rubella virus, Sabia virus, Salmonella spp., Sarcoptes scabiei, SARS coronavirus, Schistosoma spp., Shigella spp., Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus spp., Staphylococcus spp., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia spp., Taeniasolium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Variola major or minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0209] In particular examples, the relevant antigen may be derived from a pathogen selected from: severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-1 and SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), malaria parasite, Staphylococcus aureus, dengue virus, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus, and yellow fever virus.

[0210] In some embodiments, the relevant pathogenic antigen may be selected from the following: outer membrane protein A OmpA, biofilm-associated protein Bap, transport protein MucK (Acinetobacter baumannii, Acinetobacter infection); variable surface glycoprotein VSG, microtubule-associated protein MAPP15, trans-sialidase TSA (Trypanosoma brucei, African trypanosomiasis); HIV p24 antigen, HIV envelope proteins (Gp120, Gp41, Gp160), polyprotein GAG, negative factor protein Nef, transactivator of transcription Tat (HIV (human immunodeficiency virus), AIDS (acquired immune deficiency syndrome)); galactose-inhibitory adhesion protein GIAP, 29 kDa antigen Eh29, Gal / GalNAc lectin, protein CRT, 125 kDa immunodominant antigen, protein M17, adhesin ADH112, protein STIRP (Entamoeba histolytica, Amoebiasis; major surface proteins 1-5 (MSP1a, MSP1b, MSP2, MSP3, MSP4, MSP5), type IV secretion system proteins (VirB2, VirB7, VirBll, VirD4) (Anaplasma genus, Anaplasmosis); protective antigen PA, edema factor EF, lethal factor LF, S-layer homologous protein SLH (Bacillus anthracis, Bacillus anthracis); acranolisin, phospholipase D, collagen-binding protein CbpA (Area nobacteri urn haemolyticum, Area nobacteri urn haemolyticum infection); nucleocapsid protein NP, glycoprotein precursor GPC, glycoprotein GP1, glycoprotein GP2 (Junín virus, Argentine hemorrhagic fever); chitin-protein layer protein, 14 kDa surface antigen A14, major sperm protein MSP, MSP polymerization-assembly protein MPOP, MSP fiber protein 2 MFP2, MSP polymerization-activating kinase MPAK, ABA-1-like protein ALB, protein ABA-1, cuticulin CUT-1 (Ascaris lumbricoides, Ascariasis);41 kDa allergen Asp vl3, allergen Asp f3, major conidial surface protein rodlet A, protease Peplp, GPI-anchored protein Gellp, GPI-anchored protein Crflp (genus Aspergillus, aspergillosis); family VP26 protein, VP29 protein (Astroviridae, astrovirus infection); rhoptry-related protein 1 RAP-1, merozoite surface antigen MSA-1, MSA-2 (a1, a2, b, c), 12D3, 11C5, 21B4, P29, variant erythrocyte surface antigen VESA1, apical membrane antigen 1AMA-1 (Babesia genus, babesiosis); hemolysin, enterotoxin C, PXO1-51, glycolate oxidase, ABC-transporter, penicillin-binding protein, zinc transporter family protein, pseudouridine synthetase Rsu, plasmid replication protein RepX, oligoendopeptidase F, prophage membrane protein, protein HemK, flagellar antigen H, 28.5 kDa cell surface antigen (Bacillus cereus, Bacillus cereus infection); large T antigen LT, small T antigen, capsid protein VP1, capsid protein VP2 (BK virus, BK virus infection); 29 kDa protein, caspase-3-like antigen, glycoprotein (Blastocysts hominis, Blastocystis hominis infection); yeast surface adhesin WI-1 (Blastomyces dermatitidis, blastomycosis); nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP (Machupo virus, Bolivian hemorrhagic fever); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin-binding protein A DbpA, decorin-binding protein B DbpB, flagellar filament 41-kDa core protein Fla, basement membrane protein A precursor BmpA (immunodominant antigen P39), outer surface 22-kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE (Borrelia genus, B Borrelia infections);Botulinum neurotoxins BoNT / Al, BoNT / A2, BoNT / A3, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, recombinant botulinum toxin F He domain FHc (Clostridium botulinum, botulism (and infant botulism)); nucleocapsid, glycoprotein precursor (Sabia virus, Brazilian hemorrhagic fever); copper / zinc superoxide dismutase SodC, bacterioferritin Bfr, ​​50S ribosomal protein RpIL, OmpA-like transmembrane domain-containing protein Omp31, immunogenic 39 kDa protein M5 P39, zinc ABC transporter periplasmic zinc-binding protein znuA, periplasmic immunogenic protein Bp26, 30S ribosomal protein S12 RpsL, glyceraldehyde-3-phosphate dehydrogenase Gap, 25 kDa outer membrane immunogenic protein precursor Omp25, invasion protein B lalB, trigger factor Tig, molecular chaperone DnaK, putative peptidyl-prolyl cis-trans isomerase SurA, lipoprotein Ompl9, outer membrane protein MotY Ompl6, conserved outer membrane protein D15, malate dehydrogenase Mdh, component of type IV secretion system (T4SS) VirJ, lipoprotein BAB1_0187 of unknown function (Brucella genus, brucellosis); transporter family members (LolC, OppA, and PotF), putative lipoprotein release system transmembrane protein LolC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA-encoded protein, boaB-encoded protein (Burkholderia cepacia and other Burkholderia species, Burkholderia infection); mycolyltransferase Ag85A, heat shock protein Hsp65, protein TB10.4, 19 kDa antigen, protein PstS3, heat shock protein Hsp70 (Mycobacterium ulcerans, Buruli ulcer);Norovirus major and minor viral capsid proteins VP1 and VP2, genome polyprotein, Sapovirus capsid protein VP1, protein Vp3, genome polyprotein (Caliciviridae family, Calicivirus infection (Norovirus and Sapovirus)); major outer membrane protein PorA, flagellin FlaA, surface antigen CjaA, fibronectin-binding protein CadF, aspartate / glutamate-binding ABC transporter protein PeblA, protein FspAl, protein FspA2 (Campylobacter genus, Campylobacter infection); glycolytic enzyme enolase, secreted aspartyl proteinase SAPl-10, glycophosphatidylinositol (GPI)-linked cell wall protein, protein Hyr1, complement receptor 3-related protein CR3-RP, adhesin Als3p, heat shock protein 90 kDa hsp90, cell surface hydrophobic protein CSH (usually Candida albicans and other Candida species, candidiasis); 17 kDa antigen, protein P26, trimeric autotransporter adhesin TAA, Bartonella adhesin A BadA, variably expressed outer membrane protein Vomps, protein Pap3, protein HbpA, envelope-associated protease HtrA, protein OMP89, protein GroEL, protein LalB, protein OMP43, dihydrolipoamide succinyltransferase SucB (Bartonella henselae, cat scratch disease); amastigotes surface protein-2, amastigotes-specific surface protein SSP4, cruzipain, trans-sialidase TS, trypomastigotes surface glycoprotein TSA-1, complement regulatory protein CRP-10, protein G4, protein G2, hair shaft protein PAR2, accessory flagellar rod component Pari, mucin-associated surface protein MPSP (Trypanosoma cruzi, Chagas disease (American trypanosomiasis); envelope glycoproteins (gB, gC, gE, gH, gl, gK, gL), (varicella-zoster virus (VZV), chickenpox);Major outer membrane protein MOMP, putative outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock protein Hsp60 HSP10, protein IncA, proteins from the type III secretion system, ribonucleotide reductase small chain protein NrdB, plasmid protein Pgp3, chlamydial outer membrane protein N CopN, antigen CT521, antigen CT425, antigen CT043, antigen TC0052, antigen TC0189, antigen TC0582, antigen TC0660, antigen TC0726, antigen TC0816, antigen TC0828 (Chlamydia trachomatis); low calcium response protein E LCrE, chlamydial outer membrane protein N CopN, serine / threonine-protein kinase PknD, acyl-carrier-protein S-malonyltransferase FabD, single-stranded DNA-binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, polymorphic membrane protein family (Pmp1, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, Pmp10, Pmp11, Pmp12, Pmp13, Pmp14, Pmp15, Pmp16, Pmp17, Pmp18, Pmp19, Pmp20, Pmp21), (Chlamydophila pneumoniae, Chlamydophila pneumoniae infection); cholera toxin B CTB, toxin coregulatory pyrin A TcpA, toxin coregulatory pyrin TcpF, toxin coregulatory pyrin biosynthesis protein F TcpF, cholera enterotoxin subunit A, cholera enterotoxin subunit B, heat-stable enterotoxin ST, mannose-sensitive hemagglutinin MSHA, outer membrane protein U porin ompU, porin B protein, polymorphic membrane protein D (Vibrio cholerae, Cholera); propionyl-CoA carboxylase PCC, 14-3-3 protein, prohibitin, cysteine ​​protease, glutathione transferase, gelsolin, cathepsin L proteinase CatL, coat protein 20.8kDa TP20.8, coat protein 31.8kDa TP31.8, lysophosphatidic acid phosphatase LPAP, (Clonorchis sinensis, Clonorchiasis);Surface layer protein SLP, glutamate dehydrogenase antigen GDH, toxin A, toxin B, cysteine ​​protease Cwp84, cysteine ​​protease Cwpl3, cysteine ​​protease Cwpl9, cell wall protein CwpV, flagellar protein FliC, flagellar protein FliD (Clostridium difficile, Clostridium difficile infection); rhinovirus: capsid proteins VP1, VP2, VP3, VP4; coronaviruses: spike protein S, envelope protein E, membrane protein M, nucleocapsid protein N (usually rhinoviruses and coronaviruses, common cold (acute viral nasopharyngitis; acute coryza)); prion protein Prp (CJD prion, Creutzfeldt-Jakob disease; Martijn-Jakob disease (CJD)); envelope protein Gc, envelope protein Gn, nucleocapsid protein (Crimean-Congo hemorrhagic fever virus, Crimean-Congo hemorrhagic fever (CCHF)); virulence-associated DEAD-box RNA helicase VAD1, galactoxylomannan-protein GalXM, glucuronoxylomannan GXM, mannoprotein MP (Cryptococcus neoformans, cryptococcosis); acidic ribosomal protein P2 CpP2, mucin antigens Mucl, Muc2, Muc3, Muc4, Muc5, Muc6, Muc7, surface adhesion protein CP20, surface adhesion protein CP23, surface protein CP12, surface protein CP21, surface protein CP40, surface protein CP60, surface protein CP15, surface-associated glycopeptide gp40, surface-associated glycopeptide gpl5, oocyst wall protein AB, profilin PRF, apyrase (Cryptosporidium spp., cryptosporidiosis); fatty acid and retinol binding protein-1 FAR-1, tissue inhibitor of metalloproteinase TIMP (TMP), cysteine ​​proteinase ACEY-1, cysteine ​​proteinase ACCP-1, surface antigen Ac-16, secreted protein 2 ASP-2, metalloproteinase 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, adult-specific secreted factor Xa serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1 (usually Ancylostoma braziliense; several other parasites, cutaneous larva migrans (CLM); cathepsin L-like protease, 53 / 25 kDa antigen, 8 kDa family member, cysticercal protein with little trypsin-like activity TsAg5, oncocysticercal protein TSOL18, oncocysticercal protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia solium, cysticercosis);pp65 antigen, membrane protein ppl5, capsid-proximal tegument protein ppl50, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glycoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99 (cytomegalovirus (CMV), cytomegalovirus infection); capsid protein C, pre-membrane protein prM, membrane protein M, envelope protein E (domain I, domain II, domain II), protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A, protein 2K, protein NS4B, protein NS5 (dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4) - flavivirus, dengue fever); 39 kDa protein (Dientamoeba fragilis, diphtheria toxin precursor Tox, diphtheria toxin DT, pilin-specific sortase SrtA, shaft pilin protein SpaA, tip pilin protein SpaC, minor pilin protein SpaB, surface-associated protein DIP1281 (Corynebacterium diphtheriae, diphtheriae); glycoprotein GP, ​​nucleoprotein NP, minor matrix protein VP24, major matrix protein VP40, transcription activator VP30, polymerase cofactor VP35, RNA polymerase L (Ebola virus (EBOV), Ebola hemorrhagic fever); prion protein (vQD prion, variant Creutzfeldt-Jakob disease (vCJD, nvCJD)); UvrABC system protein B, protein Flpl, protein Flp2, protein Flp3, protein TadA, hemoglobin receptor HgbA, outer membrane protein TdhA, protein CpsRA, regulator CpxR, protein SapA, 18kDa antigen, outer membrane protein NcaA, protein LspA, protein LspAl, protein LspA2, protein LspB, outer membrane component DsrA, lectin DltA, lipoprotein Hip, major outer membrane protein OMP, outer membrane protein OmpA2 (Haemophilus ducreyi, soft chancre);Aspartyl protease 1 Pepl, phospholipase B PLB, alpha-mannosidase 1 AMN1, glucanosyltransferase GEL1, urease URE, peroxisomal matrix protein Pmpl, proline-rich antigen Pra, human T cell reactive protein TcrP (Coccidioides immitis and Coccidioides posadasii, coccidioidomycosis); allergen Tri r 2, heat shock protein 60 Hsp60, fungal actin Act, antigen Tri r2, antigen Tri r4, antigen Tri tl, protein IV, glycerol-3-phosphate dehydrogenase Gpdl, osmosensor HwSholA, osmosensor HwSholB, histidine kinase HwHhk7B, allergen Mala s 1, allergen Mala s 11, thioredoxin Trx Mala s 13, allergen Mala f, allergen Mala s (usually Trichophyton spp., Epidermophyton spp., Malassezia spp., Hortaea werneckii, dermatophytosis); protein EG95, protein EG10, protein EG18, protein EgA31, protein EM18, antigen EPC1, antigen B, antigen 5, protein P29, protein 14-3-3, 8 kDa protein, myophilin, heat shock protein 20 HSP20, glycoprotein GP-89, fatty acid binding protein FAPB (Echinococcus spp., echinococcosis); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigen protein MAPI, major antigen protein MAP1-2, major antigen protein MAP1B, major antigen protein MAP1-3, Erum2510-encoded protein, protein GroEL, protein GroES, 30-kDa major outer membrane protein, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa protein (Ehrlichia genus, ehrlichiosis);Secreted antigen SagA, sagA-like proteins SalA and SalB, collagen adhesin Scm, surface proteins Fmsl (EbpA(fm), Fms5 (EbpB(fm), Fms9 (EpbC(fm) and FmslO), protein EbpC(fm), 96 kDa immune defense glycoprotein Gl (Enterococcus spp., enterococcal infection); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (Enterovirus spp., enteric virus infection); outer membrane protein OM, 60 kDa outer membrane protein, cell surface antigen OmpA, cell surface antigen OmpB (sca5), 134 kDa Outer membrane protein, 31-kDa outer membrane protein, 29.5-kDa outer membrane protein, cell surface protein SCA4, cell surface protein Adr1 (RP827), cell surface protein Adr2 (RP828), cell surface protein SCA1, invasion protein invA, cell division protein fts, secreted proteins secO family, virulence proteins virB, tlyA, tlyC, parvulin-like protein Pip, preprotein translocase SecA, 120-kDa surface protein antigen SPA, 138-kD complex antigen, major 100-kD protein (protein I), cytoplasmic protein D, protective surface protein antigen SPA (Rickettsia prowazekii, typhus; Epstein-Barr nuclear antigens (EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP)), latent membrane proteins (LMP-1, LMP-2A, LMP-2B), early antigen EBV-EA, membrane antigen EBV-MA, viral capsid antigen EBV-VCA, alkaline nuclease EBV-AN, glycoprotein H, glycoprotein gp350, glycoprotein gp110, glycoprotein gp42, glycoprotein gHgL, glycoprotein gB (Epstein-Barr virus (EBV), Epstein-Barr virus infectious mononucleosis); capsid protein VP2, capsid protein VP1, major protein NS1 (parvovirus B19, erythema infectiosum (fifth disease));pp65 antigen, glycoprotein 105, major capsid protein, envelope glycoprotein H, protein U51 (human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), exanthem subitum); thioredoxin-glutathione reductase TGR, cathepsins L1 and L2, Kunitz-type protein KTM, leucine aminopeptidase LAP, cysteine ​​proteinase Fas2, saposin-like protein-2 SAP-2, thioredoxin peroxidase TPx, Prx-1, Prx-2, cathepsin I cysteine ​​proteinase CL3, cathepsin L protease CLl, phosphoglycerate kinase PGK, 27 kDa secretory protein, 60 kDa protein HSP35 alpha, glutathione transferase GST, 28.5 kDa envelope antigen 28.5 kDa TA, cathepsin B3 protease CatB3, type I cystatin stefin-1, cathepsin L5, cathepsin Llg, and cathepsin B, fatty acid-binding protein FABP, leucine aminopeptidase LAP (Fasciola hepatica and Fasciola gigantica, fascioliasis); prion protein (FFI prion, fatal familial insomnia (FFI)); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, hornet allergen homolog VAH, thioredoxin peroxidase 2 TPX-2, antigen protein SXP (peptides N, N1, N2, and N3), and activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, vespid allergen homolog VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen Col-4, secreted larval acidic protein SLAP, chitinase CHI-1, maltose-binding protein MBP, glycolytic enzyme fructose-l,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, onchocystatin CPI-2, Cox-2 (Filarioidea superfamily, filariasis);Phospholipase C PLC, heat-labile enterotoxin B, iota toxin component 1b, protein CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, perfringolysin 0 Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, fructose-bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha toxoid ATd, epsilon toxoid ETd, protein HP, large cytotoxin TpeL, endo-beta-N-acetylglucosaminidase Naglu, phosphoglyceride mutase Pgm (Clostridium perfringens, food poisoning caused by Clostridium perfringens); leukotoxin IktA, adhesion FadA, outer membrane protein RadD, high molecular weight arginine-binding protein (F; Fusobacterium spp., Fusobacterium infection; phospholipase C PLC, heat-labile enterotoxin B, iota toxin component 1b, protein CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, perfringolysin 0 Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, fructose-bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha toxoid ATd, epsilon-toxoid ETd, protein HP, large cytotoxin TpeL, endo-beta-N-acetylglucosaminidase Naglu, phosphoglycerin mutase Pgm (usually Clostridium perfringens; other Clostridium species, gas gangrene (Clostridial) myonecrosis); lipase A, lipase B, peroxidase Decl (Geotrichum candidum, geotrichosis); prion protein (GSS prion, Gerstmann-Straussler-Scheiker syndrome (GSS)); cyst wall proteins CWP1, CWP2, CWP3, modified surface proteins VSP, VSP1, VSP2, VSP3, VSP4, VSP5, VSP6, 56 kDa antigen, pyruvate ferredoxin oxidoreductase PFOR, alcohol dehydrogenase E ADHE, alpha-giardin, alpha 8-giardin, alpha-giardin, beta-giardin, cysteine ​​protease, glutathione S-transferase GST, arginine deiminase ADI, fructose-1,6-bisphosphatoaldolase FBA, Giardia trophozoite antigen GTA (GTA1, GTA2), ornithine carboxyltransferase OCT, striatal fiber acebrin-like protein SALP, uridine phosphoryl-like protein UPL, alpha-tubulin, beta-tubulin (Giardia intestinalis, Giardia lambliasis);Members of the ABC transporter family (LolC, OppA, and PotF), putative lipoprotein release system transmembrane protein LolC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA-encoded protein (Burkholderia mallei, glanders); cyclophilin CyP, 24 kDa third-stage larval protein GS24, excretory-secretory products ESP (40, 80, 120, and 208 kDa) (Gnathostoma spinigerum and Gnathostoma hispidum, gnathostomiasis); pilin proteins, minor pilin-binding subunit pilC, major pilin subunit and variant pilE, pilS, phase variant protein porA, and porin B PorB, protein TraD, Neisseria outer membrane antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin-binding protein TbpA, transferrin-binding protein TbpB, PBP2, mtrR-encoded protein, ponA-encoded protein, membrane permease FbpBC, FbpABC protein system, LbpAB protein, outer membrane protein Opa, outer membrane transporter FetA, iron repression regulator MpeR (Neisseria gonorrhoeae, gonorrhea); outer membrane protein A OmpA, outer membrane protein C OmpC, outer membrane protein K17 OmpK17 (Klebsiella granulomatis, Granuloma) inguinale (Donovanosis); fibronectin-binding protein Sfb, fibronectin / fibrinogen-binding protein FBP54, fibronectin-binding protein FbaA, M protein type 1 Emml, M protein type 6 Emm6, immunoglobulin-binding protein 35 Sib35, surface protein R28 Spr28, superoxide dismutase SOD, C5a peptidase ScpA, antigen I / II Agl / II, adhesin AspA, G-related alpha2-macroglobulin-binding protein GRAB, surface fibril protein M5 (Streptococcus pyogenes, group A streptococcal infections);C protein beta antigen, arginine deiminase protein, adhesin BibA, 105 kDa protein BPS, surface antigen c, surface antigen R, surface antigen X, trypsin-resistant protein Rl, trypsin-resistant protein R3, trypsin-resistant protein R4, surface immunogenic protein Sip, surface protein Rib, leucine-rich repeat protein LrrG, serine-rich repeat protein Srr-2, C protein alpha-antigen Bca, beta antigen Bag, surface antigen epsilon, alpha-like protein ALP1, alpha-like protein ALP5, surface antigen delta, alpha-like protein ALP2, alpha-like protein ALP3, alpha-like protein ALP4, C beta protein Bac (Streptococcus agalactiae, group B streptococcal infection); transferrin-binding protein 2 Tbp2, phosphatase P4, outer membrane protein P6, peptidoglycan-associated lipoprotein Pal, protein D, protein E, adhesion and penetration protein Hap, outer membrane protein 26 Omp26, outer membrane protein P5 (fimbrin), outer membrane protein D15, outer membrane protein OmpP2, 5'-nucleotidase NucA, outer membrane protein PI, outer membrane protein P2, outer membrane lipoprotein Pep, lipoprotein E, outer membrane protein P4, fuculokinase FucK, [Cu,Zn]-superoxide dismutase SodC, protease HtrA, protein O145, alpha-galactosylceramide (Haemophilus influenzae, Haemophilus influenza infection); polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (enteroviruses, mainly Coxsackie A virus and enterovirus 71 (EV71), hand, foot, and mouth disease (HFMD)); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein G1, nucleoprotein NP, protein N, polyprotein M (Sin Nombre virus, hantavirus, hantavirus pulmonary syndrome (HPS));Heat shock protein HspA, heat shock protein HspB, citrate synthetase GltA, protein UreB, heat shock protein Hsp60, neutrophil-activating protein NAP, catalase KatA, vacuolating cytotoxin VacA, urease alpha UreA, urease beta Ureb, protein CpnlO, protein groES, heat shock protein HsplO, protein MopB, cytotoxicity-associated 10 kDa protein CAG, 36 kDa antigen, beta-lactamase HcpA, beta-lactamase HcpB (Helicobacter pylori, Helicobacter pylori infection); integral membrane protein, aggregation-prone protein, O-antigen, toxin-antigen Stx2B, toxin-antigen StxlB, adhesion-antigen fragment Int28, protein EspA, protein EspB, intimin, protein Tir, protein IntC300, protein Eae (Escherichia coli O157:H7, O111 and O104:H4, hemolytic uremic syndrome (HUS); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein Gl, nucleoprotein NP, protein N, polyprotein M (family Bunyaviridae, hemorrhagic fever with renal syndrome (HFRS)); glycoprotein G, matrix protein M, nucleoprotein N, fusion protein F, polymerase L, protein W, protein C, phosphoprotein p, nonstructural protein V (Henipavirus (Hendra virus Nipah virus), Henipavirus infection); polyprotein, glycoprotein Protein Gp2, hepatitis A surface antigen HBAg, protein 2A, viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4, protein P1B, protein P2A, protein P3AB, protein P3D (hepatitis A virus, hepatitis A); hepatitis B surface antigen HBsAg, hepatitis B core antigen HbcAg, polymerase, protein Hbx, preS2 intermediate surface protein, surface protein L, large S protein, viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4 (hepatitis B virus (HBV), hepatitis B);Envelope glycoprotein E1 gp32 gp35, envelope glycoprotein E2 NS1 gp68 gp70, capsid protein C, core protein core, polyprotein, viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4, antigen G, protein NS3, protein NS5A (hepatitis C virus, hepatitis C); viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4, large hepatitis delta antigen, small hepatitis delta antigen (hepatitis D virus, hepatitis D); viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4, capsid protein E2 (hepatitis E virus, hepatitis E);Glycoprotein L UL1, uracil-DNA glycosylase UL2, protein UL3, protein UL4, DNA replication protein UL5, portal protein UL6, virion maturation protein UL7, DNA helicase UL8, origin-binding protein UL9, glycoprotein M UL10, protein UL11, alkaline exonuclease UL12, serine-threonine protein kinase UL13, tegument protein UL14, terminase UL15, tegument protein UL16, protein UL17, capsid protein VP23, UL18, and major capsid protein VP5. UL19, membrane protein UL20, tegument protein UL21, glycoprotein H (UL22), thymidine kinase UL23, protein UL24, protein UL25, capsid protein P40 (UL26, VP24, VP22A), glycoprotein B (UL27), ICP18.5 protein (UL28), major DNA-binding protein ICP8 (UL29), DNA polymerase UL30, nuclear matrix protein UL31, envelope glycoprotein UL32, protein UL33, inner nuclear membrane protein UL34, capsid protein VP26 (UL35), large tegument protein UL36, capsid assembly protein UL37, VP19C protein (UL38), ribonucleotide reductase (large subunit) UL39, ribonucleotide reductase (small subunit) UL40, tegument protein / virion host shutoff VHS protein (UL41), DNA polymerase processivity factor UL42, membrane protein UL43, glycoprotein C (UL44), membrane protein UL45, tegument protein VP11 / 12 (UL46), tegument protein VP13 / 14 (UL47), virion maturation protein VP16 (UL48, alpha-TIF), envelope protein UL49, dUTP diphosphatase UL50, tegument protein UL51, DNA helicase / primase complex protein UL52, glycoprotein K (UL53), transcriptional regulatory protein IE63 (ICP27, UL54), Ta; Protein UL55, protein UL56, viral replication protein ICP22 (IE68, US1), protein US2, serine / threonine-protein kinase US3, glycoprotein G (US4), glycoprotein J (US5), glycoprotein D (US6), glycoprotein I (US7), glycoprotein E (US8), tegument protein US9, capsid / tegument protein US10, Vmw21 protein (US11), ICP47 protein (IE12, US12), major transcriptional activator ICP4 (IE175, RSI), E3 ubiquitin ligase ICPO (IE110), latency-associated protein 1 LRP1, and latency-associated protein 2 LRP2, neurovirulence factor RL1 (ICP34.5), latency-associated transcript LAT (herpes simplex virus 1 and 2 (HSV-1 and HSV-2), herpes simplex); heat shock protein Hsp60, cell surface protein H1C, dipeptidyl peptidase type IV DppIV, M antigen, 70 kDa protein, 17 kDa histone-like protein (Histoplasma capsulatum, histoplasmosis); fatty acid and retinol binding protein-1 FAR-1, tissue inhibitor of metalloproteinases TIMP (TMP), cysteine ​​proteinase ACEY-1, cysteine ​​proteinase ACCP-1, surface antigen Ac-16, secreted protein 2 ASP-2, metalloproteinase 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, surface-associated antigen SAA-2, adult-specific secretory factor Xa, serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1, glutathione S-transferase GST, aspartic protease APR-1, acetylcholinesterase AChE (Ancylostoma duodenale and Necator americanus, hookworm infections); protein NS1, protein NP1, protein VP1, protein VP2, protein VP3 (human bocavirus (HBoV), human bocavirus infections);Major surface protein 2 MSP2, major surface protein 4 MSP4, MSP mutant SGV1, MSP mutant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigen protein MAP1, major antigen protein MAP1-2, major antigen protein MAP1B, major antigen protein MAP1-3, Erum2510-encoded protein, protein GroEL, protein GroES, 30 kDa major outer membrane protein, GE 100 kDa protein, GE 130 kDa protein, GE 160 kDa protein (Ehrlichia ewingii, Human ewingii ehrlichiosis); major surface proteins 1-5 (MSP1a, MSP1b, MSP2, MSP3, MSP4, MSP5), type IV secretion system proteins VirB2, VirB7, VirBll, and VirD4 (Anaplasma phagocytophilum, human granulocytic anaplasmosis (HGA); protein NS1, small hydrophobic protein NS2, SH protein, fusion protein F, glycoprotein G, matrix protein M, matrix protein M2-1, matrix protein M2-2, phosphoprotein P, nucleoprotein N, polymerase L (human metapneumovirus (hMPV), human metapneumovirus infection); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigen protein MAP1, major antigen protein MAP1-2, major antigen protein MAP1B, major antigen protein MAP1-3, Erum2510-encoded protein, protein GroEL, protein GroES, 30 kDa major outer membrane protein, GE 100 kDa protein, GE 130 kDa protein, GE 160 kDa protein (Ehrlichia chaffeensis, Human monocytic ehrlichiosis); replication protein El, regulator protein E2, protein E3, protein E4, protein E5, protein E6, protein E7, protein E8, major capsid protein LI, minor capsid protein L2 (human papillomavirus (HPV), human papillomavirus (HPV) infection);Fusion protein F, hemagglutinin-neuraminidase HN, glycoprotein G, matrix protein M, phosphoprotein P, nucleoprotein N, polymerase L (human parainfluenza virus (HPIV), human parainfluenza virus infection); hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1 protein, M2 protein, NS1 protein, NS2 protein (NEP protein: nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein, and PB2 protein (Orthomyxoviridae family, influenza virus (flu)); genome polyprotein, protein E, protein M, capsid protein C (Japanese encephalitis virus, Japanese encephalitis); RTX toxin, type IV pilus, major pilus subunit PilA, regulatory transcription factors PilS and PilR, protein sigma 54, outer membrane protein (Kingella kingae, Kingella kingae infection); prion protein (kuru prion, kuru); nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP (Lassa virus, Lassa fever); peptidoglycan-associated lipoprotein PAL, 60 kDa chaperonin Cpn60 (groEL, HspB), type IV pilin PilE, outer membrane protein MIP, major outer membrane protein Momps, zinc metalloproteinase MSP (Legionella pneumophila, Legionellosis (Pontiac fever));P4 nuclease, protein WD, ribonucleotide reductase M2, surface membrane glycoprotein Pg46, cysteine ​​proteinase CP, glucose-regulated protein 78 GRP-78, stage-specific S antigen-like protein A2, ATPase Fl, beta-tubulin, heat shock protein 70 Hsp70, KMP-11, glycoprotein GP63, protein BT1, nucleoside hydrolase NH, cell surface protein Bl, ribosomal protein Pl-like protein PI, sterol 24-c-methyltransferase SMT, LACK protein, histone HI, SPB1 protein, thiol-specific antioxidant TSA, protein antigen STI1, signal peptidase SP, histone H2B, surface antigen PSA-2, cysteine ​​proteinase b Cpb (Leishmania genus, leishmaniasis); major membrane protein I, serine-rich antigen - 45 kDa, 10 kDa capellonin GroES, HSP kDa antigen, amino-oxononanoic acid synthase AONS, protein recombinase A RecA, acetyl- / propionyl-coenzyme A carboxylase alpha, alanine racemase, 60 kDa chaperonin 2, ESAT-6-like protein EcxB (L-ESAT-6), protein Lsr2, protein ML0276, heparin-binding hemagglutinin HBHA, heat shock protein 65 Hsp65, mycPl or ML0041-encoded protein, htrA2 or ML0176-encoded protein, htrA4 or ML2659-encoded protein, gcp or ML0379-encoded protein, clpC or ML0235-encoded protein (Mycobacterium leprae and Mycobacterium lepromatosis, leprosy);Outer membrane protein LipL32, membrane protein LIC10258, membrane protein LP30, membrane protein LIC12238, Ompa-like protein Lsa66, surface protein LigA, surface protein LigB, major outer membrane protein OmpLl, outer membrane protein LipL41, protein LigAni, surface protein LcpA, adhesion protein LipL53, outer membrane protein UpL32, surface protein Lsa63, flagellin FlaBl, membrane lipoprotein LipL21, membrane protein Protein pL40, Leptospiral surface adhesin Lsa27, outer membrane protein OmpL36, outer membrane protein OmpL37, outer membrane protein OmpL47, outer membrane protein OmpL54, acyltransferase LpxA (Leptospira genus, leptospirosis); listeriolysin O precursor Hly (LLO), invasion-associated protein lap (P60), listeriolysin regulatory protein PrfA, zinc metalloproteinase Mpl, phosphatidylinositol-specific phospholipase C PLC (PlcA, PlcB), O-acetyltransferase Oat, ABC-transporter permease Im.G_1771, adhesion protein LAP, LAP receptor Hsp60, adhesin LapB, hemolysin listeriolysin OLLO, protein ActA, internalin A InIA, protein InIB (Listeria monocytogenes, listeriosis); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin-binding protein A DbpA, decorin-binding protein B DbpB, flagellar filament 41 kDa core protein Fla, basic membrane protein A BmpA (immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE (usually Borrelia burgdorferi and other Borrelia species, Lyme disease);Venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, hornet allergen homolog VAH, thioredoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N, N1, N2, and N3), activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, vespid allergen homolog VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen Col-4, secretory larval acidic protein SLAP, chitinase CHI-1, maltose-binding protein MBP, glycolytic enzyme fructose-l,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, onchocystatin CPI-2, protein Cox-2 (Wuchereria bancrofti and Brugia malayi, lymphatic filariasis (elephantiasis)); glycoprotein GP, ​​matrix protein Z, polymerase L, nucleoprotein N (Lymphocytic choriomeningitis virus (LCMV, lymphocytic choriomeningitis); thrombospondin-related anonymous protein TRAP, SSP2 sporozoite surface protein 2, apical membrane antigen 1 AMA1, rhoptry membrane antigen RMA1, acidic-basic repeat antigen ABRA, transcellular protein PF, protein Pvs25, merozoite surface protein 1 MSP-1, merozoite surface protein 2 MSP-2, ring-infected erythrocyte surface antigen RESA, liver stage antigen 3 LSA-3, protein Eba-175, serine repeat antigen 5 SERA-5, circumsporozoite protein CS, merozoite surface protein 3 MSP3, merozoite surface protein 8 MSP8, enolase PF10, hepatocyte erythrocyte protein 17 kDa HEP17, erythrocyte membrane protein 1 EMP1, protein K beta merozoite surface protein 4 / 5 MSP 4 / 5, heat shock protein Hsp90, glutamate-rich TA; Protein GLURP, merozoite surface protein 4 MSP-4, protein STARP, circumsporozoite protein-related antigen precursor CRA (Plasmodium spp., malaria); nucleoprotein N, membrane-associated protein VP24, minor nucleoprotein VP30, polymerase cofactor VP35, polymerase L, matrix protein VP40, envelope glycoprotein GP (Marburg virus, Marburg hemorrhagic fever (MHF)); protein C, matrix protein M, phosphoprotein P, nonstructural protein V, hemagglutinin glycoprotein H, polymerase L, nucleoprotein N, fusion protein F (measles virus, measles); members of the ABC transporter family (LolC, OppA, and PotF), putative lipoprotein release system transmembrane protein LolC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial elongation factor-Tu EF-Tu, 17 kDa OmpA-like proteins, boaA-encoded proteins, boaB-encoded proteins (Burkholderia pseudomallei, melioidosis (Whitmore's disease)); pilin proteins, minor pilin-related subunit pilC, major pilin subunit and variant pilE, pilS, phase variant protein porA, porin B PorB, protein TraD, Neisseria outer membrane antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin-binding protein TbpA, transferrin-binding protein TbpB, PBP2, mtrR-encoded proteins, ponA-encoded proteins, membrane permease FbpBC, FbpABC protein system, LbpAB proteins, outer membrane protein Opa, outer membrane transporter FetA, iron-regulated regulator MpeR, factor H-binding protein fHbp, adhesin NadA, protein NhbA, repressor FarR (Neisseria meningitidis, meningococcal disease); 66 kDa protein, 22 kDa protein (usually Metagonimus yokagawai, metagonimosis);Polar tube proteins (34, 75, and 170 kDa (Glugea), 35, 55, and 150 kDa (Encephalitozoon)), kinesin-related proteins, the largest subunit of RNA polymerase II, the similar integral membrane protein YIPA, anti-silencing protein 1, heat shock transcription factor HSF, protein kinase, thymidine kinase, NOP-2-like nucleolar protein (Microsporidia phylum, microsporidia); CASP8 and FADD-like apoptosis regulators, glutathione peroxidase GPX1, RNA helicase NPH-II NPH2, Poly(A) polymerase catalytic subunit PAPL, major envelope protein P43K, early transcription factor 70 kDa subunit VETFS, early transcription factor 82 kDa subunit VETFL, metalloendopeptidase G1 type, nucleoside triphosphatase I NPH1, replication protein A28-like MC134L, RNA polymerase 7 kDa subunit RP07 (molluscum contagiosum virus (MCV), molluscum contagiosum (MC)); matrix protein M, phosphoprotein P / V, small hydrophobic protein SH, nucleoprotein N, protein V, fusion glycoprotein F, hemagglutinin-neuraminidase HN, RNA polymerase L (mumps virus, mumps); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, cytoplasmic protein D, crystalline surface layer protein SLP, protective surface protein antigen SPA (Rickettsia typhi, scrub typhus (endemic typhus)); adhesin PI, adhesion P30, protein pll6, protein P40, cytoskeletal protein HMW1, cytoskeletal protein HMW2, cytoskeletal protein HMW3, MPN152-encoded protein, MPN426-encoded protein, MPN456-encoded protein, MPN-500-encoded protein (Mycoplasma pneumoniae, Mycoplasma pneumonia);NocA, iron-dependent regulatory protein; VapA, VapD, VapF, VapG, caseinolytic protease, filament tip-associated 43 kDa protein; protein P24; protein P61; 15 kDa protein; 56 kDa protein (usually Nocardia asteroides and other Nocardia species, Nocardiosis); venom allergen homolog-like protein VAL-1; abundant larval transcript ALT-1; abundant larval transcript ALT-2; thioredoxin peroxidase TPX; vespid allergen homolog VAH; thioredoxin peroxidase 2 TPX-2; antigenic protein SXP (peptides N, N1, N2, and N3); activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, vespid allergen homolog VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen Col-4, secretory larval acidic protein SLAP, chitinase CHI-1, maltose-binding protein MBP, glycolytic enzyme fructose-l,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, onchocystatin CPI-2, Cox-2 (Onchocerca volvulus, onchocerciasis (river blindness)); 43 kDa Secretory glycoprotein, glycoprotein gpO, glycoprotein gp75, antigen Pb27, antigen Pb40, heat shock protein Hsp65, heat shock protein Hsp70, heat shock protein Hsp90, protein P10, triosephosphate isomerase TPI, N-acetyl-glucosamine-binding lectin paracoccin, 28 kDa protein Pb28 (Paracoccidioides brasiliensis, paracoccidioidomycosis (South American blastomycosis)); 28 kDa cruzipain-like cysteine ​​protease Pw28CCP (usually Paragonimus westermani and other Paragonimus species, paragonimiasis);Outer membrane protein OmpH, outer membrane protein Omp28, protein PM1539, protein PM0355, protein PM1417, repair protein MutL, protein BcbC, protein PM0305, formate dehydrogenase-N, protein PM0698, protein PM1422, DNA gyrase, lipoprotein PlpE, adhesion protein Cp39, heme acquisition receptor HasR, 39 kDa capsular protein, iron-regulated OMP IROMP, outer membrane protein OmpA87, pilus protein Ptf, pilus subunit protein PtfA, transferrin-binding protein Tbpl, esterase enzyme MesA, Pasteurella multocida toxin PMT, adhesion protein Cp39 (Pasteurella genus, Pasteurellosis); filamentous hemagglutinin FhaB, adenylate cyclase CyaA, pertussis toxin subunit 4 precursor PtxD, pertactin precursor Prn, toxin subunit 1 PtxA, protein Cpn60, protein brkA, pertussis toxin subunit 2 precursor PtxB, pertussis toxin subunit 3 precursor PtxC, pertussis toxin subunit 5 precursor PtxE, pertactin Prn, protein Fim2, protein Fim3; (Bordetella pertussis, whooping cough) cough)));"Fl capsular antigen, virulence-associated V antigen, secreted effector protein LcrV, V antigen, outer membrane protease Pla, secreted effector protein YopD, putative secreted protein-tyrosine phosphatase Yoph, needle complex major subunit YscF, protein kinase YopO, putative autotransporter protein YapF, inner membrane ABC-transporter YbtQ (Irp7), putative carbohydrate-binding protein YPO0612, heat shock protein 90 HtpG, putative sulfatase protein YdeN, outer membrane lipoprotein carrier protein LolA, secretion chaperone YerA, putative lipoprotein YPO0420, hemolysin activator protein HpmB, pesticin / yersiniabactin outer membrane receptor Psn, secreted effector protein YopE, secreted effector protein YopF, secreted effector protein YopK, outer membrane protein YopN, outer membrane protein YopM, coagulase / fibrinolysin precursor Pla;" (Yersinia pestis, bubonic plague); protein PhpA, surface adhesin PsaA, pneumolysin Ply, ATP-dependent protease CIp, lipoate-protein ligase LplA, cell wall surface-anchored protein psrP, sortase SrtA, glutamyl-tRNA synthetase GltX, choline-binding protein A CbpA, pneumococcal surface protein A PspA, pneumococcal surface protein C PspC, 6-phosphogluconate dehydrogenase Gnd, iron-binding protein PiaA, murein hydrolase LytB, protease LytC, protease Al (Streptococcus pneumoniae, pneumococcal infection); major surface protein B, kexin protease KEX1, protein A12, 55-kDa antigen P55, major surface glycoprotein Msg (Pneumocystis jirovecii, Pneumocystis pneumonia (PCP); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (poliovirus, poliovirus infection); protein Nfal, exendin-3, secretory lipase, cathepsin B-like protease, cysteine ​​protease, cathepsin, peroxiredoxin, protein CrylAc (usually Naegleria fowleri, primary amebic meningoencephalitis (PAM)); agnoprotein, large T antigen, small T antigen, major capsid protein VP1, minor capsid protein Vp2 (JC virus, progressive multifocal leukoencephalopathy);Low calcium response protein E LCrE, chlamydial outer protein N CopN, serine / threonine-protein kinase PknD, acyl-carrier protein S-malonyltransferase FabD, single-stranded DNA-binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, polymorphic membrane protein family (Pmp1, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, PmplO, Pmpll, Pmpl2, Pmpl3, Pmpl4, Pmpl5, Pmpl6, Pmpl7, Pmpl8, Pmpl9, Pmp20, Pmp21) (Chlamydophila psittaci, psittacosis); outer membrane protein PI, heat shock protein B HspB, peptide ABC transporter, GTP-binding protein, protein IcmB, ribonuclease R, phosphatase SixA, protein DsbD, outer membrane protein TolC, DNA-binding protein PhoB, ATPase DotB, heat shock protein B; HspB, membrane protein ComI, 28 kDa protein, DNA-3-methyladenine glycosidase I, outer membrane protein OmpH, outer membrane protein AdaA, glycine cleavage system T-protein (Coxiella burnetii, Q fever); nucleoprotein N, large structural protein L, phosphoprotein P, matrix protein M, glycoprotein G (rabies virus, rabies); fusion protein F, nucleoprotein N, matrix protein M, matriprotein; cytoplasmic protein M2-1, matrix protein M2-2, phosphoprotein P, small hydrophobic protein SH, major surface glycoprotein G, polymerase L, nonstructural protein 1 NS1, nonstructural protein 2 NS2 (respiratory syncytial virus (RSV), respiratory syncytial virus infection); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (rhinovirus, rhinovirus infection); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, protein PS120, cytoplasmic protein D, protective surface protein antigen SPA (Rickettsia genus, Rickettsia infection); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, cytoplasmic protein D (Rickettsia akari, Rickettsialpox); envelope glycoprotein GP, ​​polymerase L, nucleoprotein N, nonstructural protein NS (Rift Valley fever virus, Rift Valley fever (RVF)); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, cytoplasmic protein D (Rickettsia rickettsii, Rocky Mountain spotted fever (RMSF)); nonstructural protein 6 NS6, nonstructural protein 2 NS2, intermediate capsid protein VP6, inner capsid protein VP2, nonstructural protein 3 NS3, RNA-directed RNA polymerase L, protein VP3, nonstructural protein 1 NS1, nonstructural protein 5 NS5, outer capsid glycoprotein VP7, nonstructural glycoprotein 4 NS4, outer capsid protein VP4; (rotavirus, rotavirus infection); polyprotein P200, glycoprotein E1, glycoprotein E2, protein NS2, capsid protein C (rubella virus, rubella);Chaperonin GroEL (MopA), inositol phosphate phosphatase SopB, heat shock protein HsIU, chaperone protein DnaJ, protein TviB, protein IroN, flagellin FliC, invasion protein SipC, glycoprotein gp43, outer membrane protein LamB, outer membrane protein PagC, outer membrane protein TolC, outer membrane protein NmpC, outer membrane protein FadL, transport protein SadA, transferase WgaP, effector proteins SifA, SteC, SseL, SseJ and SseF (Sa Salmonella genus, salmonellosis); protein 14, nonstructural protein NS7b, nonstructural protein NS8a, protein 9b, protein 3a, nucleoprotein N, nonstructural protein NS3b, nonstructural protein NS6, protein 7a, nonstructural protein NS8b, membrane protein M, envelope small membrane protein EsM, replicase polyprotein 1a, spike glycoprotein S, replicase polyprotein 1ab; SARS coronavirus, SARS (severe acute respiratory syndrome); serine protease, atypical scabies antigen 1 ASA1, glutathione S-transferase GST, cysteine ​​protease, serine protease, apolipoprotein (Sarcoptes scabiei, scabies); glutathione S-transferase GST, paramyosin, hemoglobinase SM32, major egg antigen, 14 kDa fatty acid-binding protein Sml4, major larval surface antigen P37, 22.6 kDa tegument antigen, calpain CANP, triphosphate isomerase Tim, surface protein 9B, outer capsid protein VP2, 23 kDa integral membrane protein Sm23, Cu / Zn-superoxide dismutase, glycoprotein Gp, myosin (Schistosoma spp., schistosomiasis (bilharzia)); 60 kDa chaperonin, 56 kDa type-specific antigen, pyruvate phosphate dikinase, 4-hydroxybenzoate octaprenyltransferase (Orientia tsutsugamushi, scrub typhus);Dehydrogenase GuaB, invasion protein Spa32, invasin IpaA, invasin IpaB, invasin IpaC, invasin IpaD, invasin IpaH, invasin IpaJ (Shigella genus, shigellosis (bacillary dysentery)); protein P53, virion protein US10 homolog, transcriptional regulator IE63, transcriptional transactivator IE62, protease P33, alpha transinducible factor 74 kDa protein, deoxyuridine 5'-triphosphate nucleotidyl hydrolase, transcriptional transactivator IE4, membrane protein UL43 homolog, nuclear Phosphoprotein UL3 homolog, nucleoprotein UL4 homolog, origin-binding protein, membrane protein 2, phosphoprotein 32, protein 57, DNA polymerase processivity factor, portal protein 54, DNA primase, tegument protein UL14 homolog, tegument protein UL21 homolog, tegument protein UL55 homolog, tripartite terminase subunit UL33 homolog, tripartite terminase subunit UL15 homolog, capsid-associated protein 44, virion-packaging protein 43 (Varicella zoster virus (VZV), Herpes zoster (Herpes zoster); truncated 3-beta-hydroxy-5-enesteroid dehydrogenase homolog, virion membrane protein A13, protein A19, protein A31, truncated protein A35 homolog, protein A37.5 homolog, protein A47, protein A49, protein A51, semaphorin-like protein A43, serine proteinase inhibitor 1, serine proteinase inhibitor 2, serine proteinase inhibitor 3, protein A6, protein B15, protein CI, protein C5, protein C6, protein F7, protein F8, protein F9, protein F11, protein F14, protein F15, protein F16 (variola major or minor, smallpox); adhesin / glycoprotein gp70, protease (Sporothrix schenckii, sporotrichosis);Heme-iron-binding protein IsdB, collagen adhesin Cna, clumping factor A ClfA, protein MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntCl, enterotoxin type C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, toxic shock syndrome toxin-1 TSST-1, staphylokinase, penicillin-binding protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus genus, staphylococcal food poisoning); heme-iron-binding protein IsdB, collagen adhesin Cna, clumping factor A ClfA, protein MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntCl, enterotoxin type C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, toxic shock syndrome toxin-1 TSST-1, staphylokinase, penicillin-binding protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus genus, e.g., aureus, Staphylococcus infection); antigen Ss-IR, antigen NIE, strongylastacin, Na+-K+ATPase Sseat-6, tropomycin SsTmy-1, protein LEC-5, 41 kDa antigen P5, 41 kDa larval protein, 31 kDa larval protein, 28 kDa larval protein (Strongyloides stercoralis, Strongyloidiasis);Glycerophosphodiesterase GlpQ (Gpd), outer membrane protein TmpB, protein Tp92, antigen TpFl, repeat protein Tpr, repeat protein F TprF, repeat protein G TprG, repeat protein I Tprl, repeat protein J TprJ, repeat protein K TprK, treponema membrane protein A TmpA, lipoprotein, 15 kDa Tppl5, 47 kDa membrane antigen, miniferritin TpFl, adhesin Tp0751, lipoprotein TP0136, protein TpN17, protein TpN47, outer membrane protein TP0136, outer membrane protein TP0155, outer membrane protein TP0326, outer membrane protein TP0483, outer membrane protein TP0956 (Treponema pallidum, syphilis); cathepsin L-like protease, 53 / 25 kDa antigen, 8 kDa family member, Cysticercus protein TsAg5 with little trypsin-like activity, oncophora protein TSOL18, oncophora protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia genus, cestode disease); tetanus toxin TetX, tetanus toxin C TTC, 140 kDa S-layer protein, flavoprotein beta-subunit CT3, phospholipase (lecithinase), phosphocarrier protein HPr (Clostridium tetani, tetanus); genomic polyprotein, protein E, protein M, capsid protein C (tick-borne encephalitis virus (TBEV), tick-borne encephalitis); 58 kDa antigen, 68 kDa antigen, Toxocara larvae excretory-secretory antigen TES, 32 kDa glycoprotein, glycoprotein TES-70, glycoprotein GP31, excretory-secretory antigen TcES-57, periintestinal antigen Pe, soluble extractable antigen Ex, excretory / secretory larval antigen ES, antigen TES-120, polyprotein allergen TBA-1, cathepsin L-like cysteine ​​protease c-cpl-1, 26 kDa protein (Toxocara canis or Toxocara cati, toxocariasis (ocular larva migrans (OLM) and visceral larva migrans (VLM)));Short proteins (MICl, MIC2, MIC3, MIC4, MIC5, MIC6, MIC7, MIC8), rhoptry protein Rop2, rhoptry proteins (Ropl, Rop2, Rop3, Rop4, Rop5, Rop6, Rop7, Rop16, Rjopl7), protein SR1, surface antigen P22, major antigen p24, major surface antigen p30, dense granule proteins (GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA9, GRA10), 28 kDa antigen, surface antigen SAG1, SAG2-related antigen, nucleoside-triphosphatase 1, nucleoside-triphosphatase 2, protein Stt3, HesB-like domain-containing protein, rhomboid-like protease 5, toxomepsin 1 (Toxoplasma gondii, toxoplasmosis); 43 kDa secreted glycoprotein, 53 kDa secreted glycoprotein, paramyosin, antigen Ts21, antigen Ts87, antigen p46000, TSL-1 antigen, caveolin-1 CAV-1, 49 kDa newborn larval antigen, prosaposin homolog, serine protease, serine proteinase inhibitor, 45 kDa glycoprotein Gp45 (Trichinella spiralis, trichinosis); Myb-like transcription factors (Myb1, Myb2, Myb3), adhesion protein AP23, adhesion protein AP33, adhesion protein AP33-3, adhesion AP51, adhesion AP65, adhesion protein AP65-1, alpha-actinin, kinesin-related protein, teneurin, 62 kDa proteinase, subtilisin-like serine protease SUB1, cysteine ​​proteinase gene 3 CP3, alpha-enolase En; ol, cysteine ​​proteinase CP30, heat shock proteins (Hsp70, Hsp60), immunogenic protein P270 (Trichomonas vaginalis, trichomoniasis); beta-tubulin, 47 kDa protein, secretory leukocyte-like proteinase-1 SLP-1, 50 kDa protein TT50, 17 kDa antigen, 43 / 47 kDa protein (Trichuris trichiura, trichuriasis (whipworm infection)); protein ESAT-6 (EsxA), 10 kDa filtrate antigen EsxB, secretory antigen 85-B FBPB, fibronectin-binding protein A FbpA (Ag85A), serine protease PepA, PPE family protein PPE18, fibronectin-binding protein D FbpD, immunogenic protein MPT64, secretory protein MPT51, catalase-peroxidase-peroxynitritase T KATG, periplasmic phosphate-binding lipoprotein PSTS3 (PBP-3, Phos-1), iron-regulated heparin-binding hemagglutinin Hbha, PPE family protein PPE14, PPE family protein PPE68, protein Mtb72F, protein Apa, immunogenic protein MPT63, periplasmic phosphate-binding lipoprotein PSTS1 (PBP-1), molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permease protein pstA, 14 kDa antigen, fibronectin-binding protein C FbpCl, alanine dehydrogenase TB43, glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like protein, protein MTB32, protein MTB39, crystallin, heat shock protein HSP65, protein PST-S (usually Mycobacterium tuberculosis, tuberculosis);Outer membrane protein FobA, outer membrane protein FobB, intracellular growth locus IglCl, intracellular growth locus IglC2, aminotransferase Wbtl, chaperonin GroEL, 17-kDa major membrane protein TUL4, lipoprotein LpnA, chitinase family 18 protein, isocitrate dehydrogenase, Nif3 family protein, type IV pilin glycosylation protein, outer membrane protein tolC, FAD-binding family protein, type IV pilin multimeric outer membrane protein, two-component sensor protein KdpD, chaperone protein DnaK, protein TolQ (Francisella tularensis, tularemia); MB antigen, urease, protein GyrA, protein GyrB, protein ParC, protein ParE, lipid-associated membrane protein LAMP, thymidine kinase TK, phospholipase PL-A1, phospholipase PL-A2, phospholipase PL-C, surface-expressed 96-kDa antigen; (Ureaplasma urealyticum, Ureaplasma urealyticum infection); nonstructural polyprotein, structural polyprotein, capsid protein CP, protein E1, protein E2, protein E3, protease P1, protease P2, protease P3 (Venezuelan equine encephalitis virus, Venezuelan equine encephalitis); glycoprotein GP, ​​matrix protein Z, polymerase L, nucleoprotein N (Guanarito virus, Venezuelan hemorrhagic fever); polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (West Nile virus, West Nile fever); capsid protein CP, protein E1, protein E2, protein E3, protease P2 (Western equine encephalitis virus, Western equine encephalitis); genomic polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (Yellow fever virus, Yellow fever);Putative Yop targeting protein YobB, effector protein YopD, effector protein YopE, protein Yoph, effector protein YopJ, protein translocation protein YopK, effector protein YopT, protein YpkA, flagellar biosynthesis protein FlhA, peptidase M48, potassium efflux system KefA, transcriptional regulator RovA, adhesin Ifp, translocator protein LcrV, protein PcrV, invasin Inv, outer membrane protein OmpF-like porin, adhesin YadA, protein kinase C, phospholipase CI, protein PsaA, mannosyltransferase-like protein WbyK, protein YscU, antigen YPMa (Yersinia pseudotuberculosis, Yersinia pseudotuberculosis infection); effector protein YopB, 60 kDa chaperonin, protein WbcP, tyrosine protein phosphatase Yoph, protein YopQ, enterotoxin, galactoside permease, reductase NrdE, protein YasN, invasin Inv, adhesin YadA, outer membrane porin F OmpF, protein UspAl, protein EibA, protein Hia, cell surface protein Ail, chaperones SycD, protein LcrD, protein LcrG, protein LcrV, protein SycE, protein YopE, regulator protein TyeA, protein YopM, protein YopN, protein YopO, protein YopT, protein YopD, protease ClpP, protein MyfA, protein FilA, and protein PsaA (Yersinia enterocolitica, yersiniosis);

[0211] In one embodiment, the antigen is a viral antigen. For example, the viral antigen is derived from a respiratory virus. In one embodiment, the respiratory virus is selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, and bocavirus.

[0212] In one embodiment, the viral antigen is derived from influenza virus. In one embodiment, the viral antigen is derived from respiratory syncytial virus. In one embodiment, the viral antigen is derived from parainfluenza virus. In one embodiment, the viral antigen is derived from metapneumovirus. In one embodiment, the viral antigen is derived from rhinovirus. In one embodiment, the viral antigen is derived from coronavirus. In one embodiment, the viral antigen is derived from adenovirus. In one embodiment, the viral antigen is derived from bocavirus. In one embodiment, the antigen is derived from influenza virus and / or coronavirus. In one embodiment, the antigen is derived from betacoronavirus.

[0213] In embodiments where the infectious disease is influenza, the antigen may include influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2), or a fragment or variant thereof. In one embodiment, the antigen is a peptide or protein derived from influenza virus hemagglutinin (HA) and / or neuraminidase (NA), or a fragment or variant thereof. The HA and / or NA may be independently derived from influenza A virus or influenza B virus, or a fragment of either.

[0214] In one example, the antigen is derived from an influenza A virus strain. For example, the antigen is an influenza A virus hemagglutinin (HA) protein, neuraminidase (NA) protein, matrix (M) protein, nucleoprotein (NP), nonstructural (NS) protein, or an immunogenic fragment or variant thereof. In one example, the antigen is influenza A hemagglutinin (HA) subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, and / or influenza A neuraminidase (NA) subtype N1, N2, N3, N4, N5, N6, N7, N8, or N9, and / or influenza A matrix (M) protein subtype M1 or M2, and / or influenza A nonstructural (NS) protein subtype NS1 or NS2. In one embodiment, the antigen is an H5 hemagglutinin protein and / or an N1 neuraminidase protein. In one embodiment, the antigen is an M protein and / or an NP protein of an influenza A virus. For example, the antigen is an M1 matrix protein and / or an NP protein. In one embodiment, the NP protein is the A / California / 07 / 09 strain. In one embodiment, the antigen is an HA protein, an NA protein, and / or an M protein of an influenza A virus. For example, the antigen is an H5 hemagglutinin protein and / or an N1 neuraminidase protein and / or an M1 matrix protein and / or an M2 matrix protein.

[0215] In embodiments where the infection is caused by a coronavirus (e.g., SARS-CoV1 and / or SARS-CoV2), the antigen is a peptide or protein derived from the spike (S) protein or nucleocapsid (N). S and / or N can independently be derived from a variant of SARS-CoV-2 (e.g., original strain, alpha, delta, omicron) or a fragment of either. In one embodiment, the antigen is the SARS-CoV-2 N protein and / or S protein from SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020.

[0216] In one example, an immune response induced by a composition described herein has been demonstrated in a suitable model system. In one example, a protective response against an infectious disease (e.g., SARS-CoV-2 or influenza) induced by a composition described herein has been demonstrated in a suitable model system. For example, such a response may be demonstrated in an animal model, e.g., a non-human primate model (e.g., rhesus monkey) and / or a mouse model.

[0217] Screening assays Assays can be performed to assess the efficacy and potency of the lipid nanoparticle compositions described herein, including, for example, serology and immune response. Suitable methods are available to those skilled in the art, including, but not limited to, antigen expression, microneutralization assays, and antigen-specific T cell responses.

[0218] antigen expression In one example, the lipid nanoparticle composition is evaluated for expression of a gene of interest.

[0219] For example, antigen expression is detected using an antibody against the gene of interest. In one example, the number of cells positive for antigen expression is measured, for example, by fluorescence-activated cell sorting (FACS). In another example, the mean fluorescence intensity (MFI) is quantified, for example, using FACS. In a further example, a specific potency value or probability of successful transfection per unit mass of RNA is calculated.

[0220] Microneutralization assay In one example, the lipid nanoparticle composition is evaluated for antibody responses. For example, the lipid nanoparticle composition is evaluated using a microneutralization assay. Methods for performing a microneutralization assay will be apparent to those skilled in the art. In one example, the microneutralization assay is a short-form assay. In one example, a viral fluorescent focus-based microneutralization assay is performed. In another example, the microneutralization assay is a long-form assay.

[0221] Antigen-specific T cell response In one example, lipid nanoparticle compositions are evaluated for their ability to induce an antigen-specific T cell response. Methods for assessing the induction of an antigen-specific T cell response will be apparent to those skilled in the art and / or are described herein.

[0222] For example, detection of antigen-specific T cells is performed in spleen culture. Briefly, spleen cell cultures are established in T cell medium, and the cell cultures are stimulated with or without antigen peptides. In one example, antigen-specific T cell responses are determined using flow cytometry.

[0223] kit In another embodiment of the present disclosure, a kit is provided that includes a composition comprising an enriched population of LNPs produced using the methods described herein, which is useful for treating or preventing, or slowing the progression of, a disease or disorder such as those described above.

[0224] In one example, the kit includes (a) a container containing a composition comprising an enriched population of LNPs and / or a pharmaceutically acceptable carrier or diluent, and (b) a package insert containing instructions for treating or preventing or slowing the progression of a disease or disorder (e.g., COVID-19 or ARDS) in a subject.

[0225] According to this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds or contains a composition effective for a disease or disorder of the present disclosure and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a self-replicating RNA. The label or package insert indicates that the composition is used for treating an eligible subject (e.g., a subject suffering from or susceptible to influenza, influenza virus infection, SARS-CoV-2 infection, COVID-19, and / or ARDS), along with specific instructions for dosage and treatment interval, as well as any other medications provided. The kit may further include an additional container containing a pharmaceutically acceptable diluent buffer (e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution). The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0226] The invention is further disclosed in the following numbered paragraphs: 1. A method for preparing an enriched population of lipid nanoparticles (LNPs), comprising: contacting a composition comprising lipid nanoparticles and RNA with an anion exchanger under conditions such that the anion exchanger binds unencapsulated RNA; collecting the effluent to obtain the enriched population of lipid nanoparticles. 2. The method of paragraph 1, wherein the unencapsulated RNA comprises free RNA, RNA bound to the surface of the lipid nanoparticle, and / or partially encapsulated RNA. 3. The method of paragraph 1 or paragraph 2, wherein the composition comprising the lipid nanoparticles has an ionic concentration of 5 to 50 mM. 4. The method of any one of paragraphs 1 to 3, wherein the composition comprising lipid nanoparticles and RNA comprises salt at a concentration of 100 mM or less. 5. The method of any one of paragraphs 1 to 4, wherein the composition comprising the lipid nanoparticles has a conductivity of less than 15 mS / cm. 6. The method of any one of paragraphs 1 to 5, wherein the composition comprising the lipid nanoparticles further comprises a buffer selected from citrate buffer, Bis-Tris, histidine, acetate buffer, phosphate buffer, Tris buffer, and / or combinations thereof. 7. The method of paragraph 6, wherein the buffer is a Bis-Tris buffer, a Histidine buffer, or a Tris buffer. 8. The method of any one of paragraphs 1 to 7, wherein the LNP comprises an ionizable lipid and one or more of a neutral lipid, a PEGylated lipid, and a structured lipid. 9. The ionizable lipid is 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), 1,2-Dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 2-Hexyldecanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester (ALC-0315), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA or MC3), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), and 9. The method of paragraph 8, wherein the compound is selected from the group consisting of 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester. 10. The neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 10. The method of paragraph 8 or paragraph 9, wherein the glycerol-containing surfactant is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. 11. The method of any one of paragraphs 8 to 10, wherein the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, optionally PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE. 12. The method of any one of paragraphs 8 to 11, wherein the structured lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and α-tocopherol. 13. The method of paragraph 12, wherein the structured lipid is cholesterol and / or campesterol. 14. The methods of any one of paragraphs 1-13, wherein the LNP comprises a lipid component comprising about 25 mol% to about 60 mol% ionizable lipid, about 2 mol% to about 25 mol% neutral lipid, about 18.5 mol% to about 60 mol% structural lipid, and about 0.2 mol% to about 10 mol% PEGylated lipid. 15. The methods of any one of paragraphs 1-14, wherein the mRNA is a self-amplifying mRNA (sa-mRNA) or a conventional mRNA. 16. The method of any one of paragraphs 1 to 15, wherein the mRNA is greater than 500 nt in length. 17. The method of any one of paragraphs 1 to 15, wherein the RNA is 10,000 nt to 15,000 nt in length. 18. The method of any one of paragraphs 1 to 17, wherein the anion exchanger is an anion exchange resin or an anion exchange membrane. 19. The method of paragraph 18, wherein the anion exchange membrane is Mustang® Q, Sartobind® Q, Chromasorb®, Capto® Q, Q Sepharose Fast Flow (QSFF), Poros® Q, Fractogel® EMD, Natrix® Q, or Eshmuno® Q membrane. 20. The method of paragraph 19, wherein the anion exchange membrane is a Mustang® Q membrane. 21. The method of any one of paragraphs 1 to 20, further comprising eluting the unencapsulated RNA from the anion exchanger. 22. The method of any one of paragraphs 1 to 21, wherein the lipid nanoparticles have a diameter of about 30 nm to about 160 nm. 23. A composition comprising an enriched population of lipid nanoparticles produced by the method of any one of paragraphs 1 to 21. 24. The composition of paragraph 23, wherein at least 90% of the RNA is encapsulated within the LNP. 25. A concentrated lipid nanoparticle composition, comprising: (i) a plurality of lipid nanoparticles, each LNP comprising: ionizable lipids, neutral lipids, PEGylated lipids, and the plurality of lipid nanoparticles comprising structured lipids; and (ii) comprises RNA; The concentrated lipid nanoparticle composition, wherein at least 90% of the RNA is encapsulated within the LNPs. 26. A pharmaceutical composition comprising an enriched population of lipid nanoparticles produced by the method of any one of paragraphs 1-22 and a pharmaceutically acceptable carrier. 27. A pharmaceutical composition comprising the enriched population of lipid nanoparticles of paragraph 25 and a pharmaceutically acceptable carrier. 28. A method for delivering mRNA to mammalian cells, comprising administering to a subject the pharmaceutical composition described in paragraph 26 or 27, thereby contacting the cells with the lipid nanoparticles and delivering the RNA to the cells. 29. The method of paragraph 28, wherein the cells are from a human subject. 30. A method for producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with a pharmaceutical composition according to paragraph 26 or 27. 31. A method of treating a disease, disorder, or condition in a subject in need of such treatment, comprising administering to the subject a pharmaceutical composition described in paragraph 26 or 27, thereby treating the disease, disorder, or condition. 32. Use of a pharmaceutical composition according to paragraph 26 or 27 in the manufacture of a medicament for the treatment of a disease, disorder, or condition. 33. The method of paragraph 31 or the use of paragraph 32, wherein the disease, disorder, or condition is selected from the group consisting of a rare disease, an infectious disease, a cancer, a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease. 34. A vaccine comprising the composition of paragraph 23 or 24, the concentrated lipid nanoparticle composition of paragraph 25, or the pharmaceutical composition of paragraph 26 or 27. 35. The vaccine of paragraph 34, wherein the vaccine is selected from a tumor vaccine, an influenza vaccine, and a SARS vaccine including SARS-CoV-2.

[0227] The present disclosure includes the following non-limiting examples. [Example]

[0228] Example 1 - Preparation of an enriched population of lipid nanoparticles sa-RNA was prepared using in vitro transcription from a linearized plasmid template using standard methods. The sa-RNA encoded the H5 antigen from A / turkey / Turkey / 1 / 2005. The following construct was prepared: F500.3 (SEQ ID NO: 1). Briefly, a DNA template encoding a self-replicating RNA was produced in competent Escherichia coli cells transformed with the DNA plasmid. Individual bacterial colonies were isolated, and the resulting plasmid DNA was amplified in E. coli cultures. After fermentation, the plasmid DNA was isolated using a Maxiprep DNA kit and linearized by restriction digestion. The restriction enzyme was then removed using phenol / chloroform extraction and ethanol precipitation.

[0229] mRNA was generated by in vitro transcription from a linearized DNA template using T7 RNA polymerase. The DNA template was subsequently removed by DNase digestion. Enzymatic capping using VCE was performed to add Cap0, yielding functional mRNA. The resulting mRNA was purified and resuspended in nuclease-free water.

[0230] Lipid nanoparticle compositions containing RNA were prepared using ionizable cationic lipids, additional helper lipids, and the sa-RNA produced as described above.

[0231] LKY750, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG2k) were combined in a molar ratio of 40:10:48:2 in 3.2 mM ethanol. An mRNA solution was prepared at 0.025 mg / mL in 50 mM citrate buffer at pH 6. The lipid solution in ethanol was then rapidly mixed with the mRNA in citrate buffer using a staggered herringbone micromixer such as the NanoAssemblr benchtop instrument (Precision Nanosystems). The total flow rate (TFR) was 12 mL / min, and the flow rate ratio (FRR) was 2:1. This mixing ratio resulted in an 8:1 ratio of ionizable cationic lipid to RNA phosphate groups (N:P ratio) and a 37:1 lipid to RNA mass ratio. The mixed solution was diluted 10-fold with 50 mM citrate buffer at pH 6 and subjected to tangential flow filtration (TFF) using a 300 k molecular weight cut-off membrane (mPES) until concentrated to the original volume.

[0232] The citrate buffer was then replaced with a buffer containing 20 mM Tris buffer, pH 7.5, 80 mM sodium chloride, and 3% sucrose using diafiltration with 10 times the volume of fresh buffer. The LNP solution was concentrated to a volume of 5-10 mL, filtered using a 0.2 micron PES syringe filter, aliquoted into vials, and frozen at 1°C / min using a Corning® CoolCell® LX Cell Freezing Container until the sample reached -80°C. Samples were stored at -80°C until needed for further assays. Samples may be filtered through anion exchangers before or after storage at -80°C.

[0233] After thawing, the samples were filtered through anion exchange filters (Mustang Q, Pall Corporation) and the effluent was collected.

[0234] LNPs containing sa-RNA were analyzed to determine the RNA concentration before and after anion exchange filtration (Table 1). The total amount of RNA in the sample and the percent encapsulated RNA were determined using a fluorescent assay using dyes such as Ribogreen, which become luminescent upon binding to RNA. The total amount of RNA is determined by disrupting the LNPs with detergent to expose the encapsulated RNA, adding the dye, and comparing the luminescence intensity to a standard curve generated using ribosomal RNA. By omitting the detergent disruption of the LNPs, it was thought that the amount of unencapsulated RNA could be estimated in a similar manner. Using the known total amount of RNA and the known amount of unencapsulated RNA, the percent encapsulated RNA could be calculated as follows: Encapsulation rate (%) = ((RNA 合計 -RNA カプセル化されていない ) / RNA 合計 ) x 100 In the formula, RNA 合計 and RNA カプセル化されていない are the concentrations of total RNA and unencapsulated RNA, respectively. [Table 2]

[0235] Mustang filtration was observed to reduce the total amount of RNA present in the composition by 88.8%, suggesting that only 11.2% of the RNA was fully encapsulated in the LNPs before treatment with the anion exchanger.

[0236] Example 2: Encapsulation rate To assess whether the decrease in total RNA was due to loss of incompletely encapsulated RNA in the LNPs, empty LNPs were formulated as described in Example 1 by mixing the lipid mixture with 50 mM citrate buffer at pH 6. sa-RNA was then mixed with the empty LNPs at an N:P ratio of 8:1 to a final concentration of 42 μg / mL. The LNPs were not frozen. The empty LNPs and empty LNPs + RNA formulations were filtered through anion exchange filters, and the flow-through was collected. The resulting formulations were then analyzed to determine the RNA concentrations before and after anion exchange filtration (Table 2). [Table 3]

[0237] From Table 2, it is clear that the Ribogreen assay neither measures the RNA encapsulation rate in the LNPs nor accurately reports the RNA concentration. Furthermore, the anion exchanger appears to remove RNA complexed to the outside of the LNPs.

[0238] Example 3: In vitro activity and efficacy of concentrated LNPs The ability of RNA-LNPs to transfect cultured cells before and after treatment with an anion exchanger was characterized using an in vitro assay that quantifies the percentage of cells expressing the antigen of interest. RNA-LNPs were prepared as described in Example 1, except that the RNA contained in the RNA-LNPs was sa-mRNA encoding the HA and NA subtypes from A / turkey / Turkey / 1 / 2005. The following sa-mRNA construct, described in WO2022 / 118226, was prepared: NSP1-4.SGP.H5.SGPv2.N1 (F602; also referred to as SEQ ID NO: 2).

[0239] Two-fold serial dilutions of LNP-formulated sa-RNA, prepared as described in Example 2, were electroporated or transfected into a baby hamster kidney (BHK) cell line. The antigen encoded by the sa-RNA was A / turkey / Turkey / 05 (H5-sgpv2-N1). After 17–19 hours, cells were harvested and stained for either S or N antigen expression using anti-S or anti-N antibodies. The number of cells positive for antigen expression and mean fluorescence intensity (MFI) were measured by AF4-MALS and fluorescence-activated cell sorting (FACS). The data were analyzed, and specific potency values ​​(probability of successful transfection per mass unit of RNA) were calculated. The in vitro activity and potency of LNPs before and after filtration are shown in Figure 1. Potency values ​​are based on H5+N1+ co-expression. Each LNP formulation tested exhibited 4.6–10-fold increased potency after MustangQ filtration.

[0240] Another series of test LNPs were prepared to measure in vitro activity and potency. These included SAM-H5-N1 / LNP.cholesterol, SAM-H5-N1 / LNP.campesterol, SAM-H5-N1 / LNP.cholesterol MustangQ filtered, and SAM-H5-N1 / LNP.campesterol MustangQ filtered. The antigen encoded by SAM was A / turkey / Turkey / 05 (H5-sgpv2-N1). The in vitro activity and potency of these LNP-encapsulated vaccines were determined by measuring co-expression of H5 and N1. The results are shown in Table 3 and Figure 2. Potency values ​​are based on H5+N1+ co-expression. [Table 4]

[0241] Geometric mean fluorescence intensity (GMFI) values ​​suggest higher protein expression levels per cell in Mustang-filtered LNPs compared to unfiltered LNPs. Campesterol-containing LNPs were also observed to exhibit slightly higher levels of protein expression than cholesterol-containing LNPs.

[0242] Example 4: Concentrated LNPs induce cell-mediated immune responses The ability of the concentrated LNP composition to act as a vaccine was evaluated by measuring antibody and cell-based immune responses following a prime-boost vaccination schedule. Primary vaccination was administered by intramuscular injection (i.m.) on day 0, followed by a booster vaccination 21 days later. BALB / c female mice were vaccinated with either SAM-H5-N1 / LNP.cholesterol, SAM-H5-N1 / LNP.campesterol, SAM-H5-N1 / LNP.cholesterol, Mustang-filtered, SAM-H5-N1 / LNP.campesterol, Mustang-filtered, or H5N1 subunit + MF59. The SAM-encoded antigen was A / turkey / Turkey / 05 (H5-sgpv2-N1). Each group contained 10 mice. Mice received two doses of vaccine containing either 1, 0.1, 0.01, or 0.001 μg of RNA. The H5N1 subunit + MF59 vaccine contained 1 μg of H5 protein (SRID). Mice were bled the day before the first vaccination, 21 days after the first vaccination (day 20), and again 21 days after the second vaccination (day 42).

[0243] Serum was collected at the end of the study (i.e., 42 days after the first vaccine administration or 21 days after the last second vaccine dose) to assess antibody responses. Serology of filtered and unfiltered LNPs was also assessed in hemagglutination inhibition (HAI) assays, pseudotyped virus microneutralization assays, and anti-NA inhibition assays. All constructs induced virus-specific antibodies.

[0244] For all serological assays, serum was treated with Vibrio cholerae neuraminidase (also known as receptor-destroying enzyme (RDE)) (Denka Seiken Co. Ltd., Tokyo, Japan) in the same manner and diluted to a starting dilution of 1:10 in PBS. Sheep serum against H5N1 virus (FDA / CBER Kensington lot number H5-Ag-1115) was used as a positive control serum.

[0245] Total IgG levels were assessed by ELISA (Figure 3 and Table 4). [Table 5]

[0246] The results of the HAI assay are shown in FIG. [Table 6]

[0247] The results of the pseudotype virus microneutralization assay are shown in FIG. 5 and Table 6. [Table 7]

[0248] The results of the microneutralization assays for the short and long forms are shown in Figures 6A and 6B and Tables 7 and 8, respectively. [Table 8] [Table 9]

[0249] The results of the ELLA assay (to measure NA inhibition) are shown in FIG. [Table 10]

[0250] No differences were observed between LNPs containing cholesterol and campesterol. Overall (Figure 8), filtered LNPs performed differently than unfiltered LNPs in all assays except ELLA (Figures 3-7). At low doses (e.g., 0.001 μg), filtered LNPs consistently performed better than unfiltered LNPs (Figure 8A). At high doses (e.g., 0.1 μg), the differences between filtered and unfiltered LNPs were inconsistent (Figure 8B).

[0251] Spleens were harvested, pooled, and assayed for antigen-specific CD4 and CD8 T cells using an in vitro antigen stimulation / intracellular cytokine immunofluorescence flow cytometry assay. The percentage of CD4 or CD8 T cells that produced cytokines (one or more of IL-2, IFN-γ, TNF-α, IL-5, and IL-13 according to Table 10) was quantified. Overall, filtration did not affect the generation of cell-mediated immune responses. Antigen-specific CD4 and CD8 T cell responses are shown in Figure 9. [Table 11]

[0252] Both formulations generated CD4 T cell responses. CD4 T cells elicited by the LNP vaccine were primarily Th0 (IL2+ and / or TNFα+, IFNg-, IL5-, IL13-) and Th1 (IFNg+, IL5-, IL13-) types, with little or no mixed responses (Figure 9A). Filtration using an anion exchanger did not alter the Th type. As shown in Figure 9B, immunization with both filtered and unfiltered LNP elicited similar CD8 T cell responses.

[0253] As shown in Figure 10, filtered LNPs performed better at lower doses, although the improvement due to filtration may plateau at a dose of 0.1 μg. We also observed that in most cases, responses from cholesterol-containing LNPs were superior to those from campesterol-containing LNPs.

[0254] Overall, LNPs filtered using an anion exchanger generated better Ab and T cell responses at lower doses.

[0255] Example 5: Microneutralization assay Microneutralization assays (short and long forms) were performed in a qualified mammalian cell line (proprietary 33016-PF MDCK (Madin-Darby Canine Kidney)).

[0256] Microneutralization Assay Short Form (MN Assay SF) A viral fluorescent focus-based microneutralization (FFAMN) assay was performed using an in-house developed protocol. RDE-treated test mouse samples and positive control sera were heat-inactivated and diluted to a starting dilution of 1:40 in PBS. A 4-fold serial dilution was performed in neutralization medium (Minimum Essential Medium D-MEM (GIBCO) supplemented with 1% BSA (Rockland; BSA-30), 100 U / mL penicillin, and 100 μg / mL streptomycin (GIBCO)) in a U-bottom 96-well plate (BD Falcon). A / turkey / Turkey / 1 / 2005 (H5N1) virus was diluted to approximately 1,000–1,500 fluorescent focus-forming units (FFU) / well (20,000–30,000 FFU / mL) in neutralization medium and added to the diluted sera at a 1:1 ratio.

[0257] After 2 hours of incubation at 37°C and 5% CO2, the mixture was inoculated into plates containing MDCK 33016-PF cells (Half Area 96-well plates; Corning) and incubated overnight at 37°C and 5% CO2 for 16–18 hours. MDCK 33016-PF cells were seeded at 3.0E4 / well (3.0E6 / plate) 6–8 hours prior to incubation in cell growth medium (D-MEM supplemented with 10% HyClone fetal bovine serum-FBS (Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin). After overnight incubation, the cells were fixed with a cold mixture of acetone and methanol before immunostaining.

[0258] Viruses were visualized by incubating the cells separately for 1 hour at room temperature with a monoclonal antibody specific for influenza A virus nucleoprotein (NP) (clone A1, A3 blend; Millipore; catalog no. MAB8251) and an Alexa Fluor 488 goat anti-mouse IgG (H+L) Ab (Invitrogen; catalog no. A11001), diluted in PBS buffer containing 0.05% Tween-20 (Sigma) and 2% BSA (Fraction V; Calbiochem, 2960, 1194C175). NP viral protein was quantified using a CTL Immunospot analyzer (Cellular Technology Limited, Shaker Heights, Cleveland, OH) with fluorescein isothiocyanate (FITC) fluorescence filters set at excitation and emission wavelengths of 482 and 536 nm. Fluorescent foci were counted using the Immunospot 7.0.12.1 professional analyzer DC software with the custom analysis module. Data were subsequently logged by the software into an Excel data analysis spreadsheet, and the 60% focus reduction endpoint was calculated from the average foci count in virus control wells (for each plate), and the 60% focus reduction neutralization titer was calculated by linear interpolation between the wells just above and below the 60% endpoint (for each sample).

[0259] Microneutralization Assay Long Form (MN Assay LF) The MN assay LF was performed using an in-house developed protocol. RDE-treated test mouse samples and positive control sera were heat-inactivated and diluted to a starting dilution of 1:40 in PBS. Two-fold serial dilutions were performed in a U-bottom 96-well plate (BD Falcon) in neutralization medium (30% spent growth medium (Irvine Scientific) and 70% infection medium (protein-free medium; 33016 MDCK PFM; GIBCO) supplemented with 100 U / mL penicillin, 100 μg / mL streptomycin (GIBCO), and 0.33 μg / mL TPCK-trypsin (TPCK-treated; tosylphenylalanyl chloromethyl ketone; Sigma)). A / turkey / Turkey / 1 / 2005 (H5N1) virus was diluted to 100 TCID (tissue culture infectious dose) per well in neutralization medium and added to the diluted sera at a 1:1 ratio. Pre-diluted serially diluted serum samples were incubated with the virus for 1 hour at 37°C and 5% CO2. In the inoculation step, plates (cell culture 96-well plates; Costar) containing MDCK 33016-PF cells (seeded the previous day at 3.0E4 / well (3.0E6 / plate) in antibiotic-free cell growth medium (Irvine Scientific)) were washed with sterile PBS and then infected with the mixture and incubated for 1 hour at 37°C and 5% CO2. The infection was stopped by aspirating the antibody / virus mixture, and the cells, washed with sterile PBS, were inoculated with neutralization medium (100 μl / well) containing two-fold serially diluted antibodies and then incubated for 5 days at 37°C and 5% CO2. In the final "readout" step, virus detection was performed by HA quantification using 0.5% turkey red blood cells (Lampire Biological Laboratories). The absence of infectivity constituted a positive neutralization reaction, indicating the presence of virus-specific antibodies in the serum sample.

[0260] Example 6: Hemagglutination Inhibition (HAI) Assay The HAI assay was performed as previously described (WHO (2011) Manual for the laboratory diagnosis and virological surveillance of influenza: WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland). Briefly, RDE-treated test mouse samples and positive control sera were heat-inactivated and diluted to a starting dilution of 1:10 in PBS. 2-fold serially diluted samples (25 μl) were incubated with an equal volume of A / turkey / Turkey / 1 / 2005 (H5N1) virus (4 hemagglutination units [HAU]) at room temperature (RT) for 30 minutes. An equal volume of 0.5% turkey red blood cells (Lampire Biological Laboratories) was then added and incubated for 30 minutes at room temperature. The HAI titer was expressed as the reciprocal of the highest dilution of sample that inhibited hemagglutination.

[0261] array [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7] [Table 12-8]

Claims

1. 1. A method for preparing an enriched population of lipid nanoparticles (LNPs), comprising: contacting a composition comprising lipid nanoparticles and RNA with an anion exchanger under conditions such that the anion exchanger binds unencapsulated RNA; collecting the effluent to obtain the enriched population of lipid nanoparticles.

2. 2. The method of claim 1, wherein the unencapsulated RNA comprises free RNA, RNA bound to the surface of the lipid nanoparticle, and / or partially encapsulated RNA.

3. The method of claim 1, wherein the composition comprising the lipid nanoparticles has an ionic concentration of 5 to 50 mM.

4. The method of claim 1, wherein the composition comprising lipid nanoparticles and RNA comprises a salt at a concentration of 100 mM or less.

5. 10. The method of claim 1, wherein the composition comprising the lipid nanoparticles has a conductivity of less than 15 mS / cm.

6. 2. The method of claim 1, wherein the composition comprising the lipid nanoparticles further comprises a buffer selected from citrate buffer, Bis-Tris, histidine, acetate buffer, phosphate buffer, Tris buffer, and / or combinations thereof.

7. 7. The method of claim 6, wherein the buffer is a Bis-Tris buffer, a Histidine buffer, or a Tris buffer.

8. 10. The method of claim 1, wherein the LNP comprises an ionizable lipid and one or more of a neutral lipid, a PEGylated lipid, and a structured lipid.

9. The ionizable lipid is 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 2-Hexyldecanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl]ester (ALC-0315), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA or MC3), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), and 9. The method of claim 8, wherein the hydroxyl group is selected from the group consisting of 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester.

10. The neutral lipid may be 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 10. The method of claim 8, wherein the glycerol-containing compound is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

11. 9. The method of claim 8, wherein the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, optionally PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

12. 9. The method of claim 8, wherein the structured lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and α-tocopherol.

13. 13. The method of claim 12, wherein the structured lipid is cholesterol and / or campesterol.

14. 10. The method of claim 1, wherein the LNPs comprise a lipid component comprising about 25 mol% to about 60 mol% ionizable lipids, about 2 mol% to about 25 mol% neutral lipids, about 18.5 mol% to about 60 mol% structured lipids, and about 0.2 mol% to about 10 mol% PEGylated lipids.

15. The method of claim 1, wherein the mRNA is a self-amplifying mRNA (sa-mRNA) or a conventional mRNA.

16. 16. The method of claim 15, wherein the mRNA is greater than 500 nt in length.

17. 2. The method of claim 1, wherein the RNA is 10,000 nt to 15,000 nt in length.

18. The method according to claim 1, wherein the anion exchanger is an anion exchange resin or an anion exchange membrane.

19. 19. The method of claim 18, wherein the anion exchange membrane is Mustang® Q, Sartobind® Q, Chromasorb®, Capto® Q, Q Sepharose Fast Flow (QSFF), Poros® Q, Fractogel® EMD, Natrix® Q, or Eshmuno® Q membrane.

20. 20. The method of claim 19, wherein the anion exchange membrane is a Mustang® Q membrane.

21. 21. The method of claim 20, further comprising eluting the unencapsulated RNA from the anion exchanger.

22. 2. The method of claim 1, wherein the lipid nanoparticles have a diameter of about 30 nm to about 160 nm.

23. 10. A composition comprising an enriched population of lipid nanoparticles produced by the method of claim 1.

24. 24. The composition of claim 23, wherein at least 90% of the RNA is encapsulated within the LNPs.

25. 1. A concentrated lipid nanoparticle composition comprising: (i) a plurality of lipid nanoparticles, each LNP comprising: ionizable lipids, neutral lipids, PEGylated lipids, and the plurality of lipid nanoparticles comprising structured lipids; and (ii) comprises RNA; The concentrated lipid nanoparticle composition, wherein at least 90% of the RNA is encapsulated within the LNPs.

26. 10. A pharmaceutical composition comprising an enriched population of lipid nanoparticles produced by the method of claim 1 and a pharmaceutically acceptable carrier.

27. 26. A pharmaceutical composition comprising the enriched population of lipid nanoparticles of claim 25 and a pharmaceutically acceptable carrier.

28. A method for delivering mRNA to mammalian cells, comprising administering the pharmaceutical composition of claim 26 to a subject, thereby contacting the cells with the lipid nanoparticles and delivering the RNA to the cells.

29. 29. The method of claim 28, wherein the cells are from a human subject.

30. 27. A method for producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the pharmaceutical composition of claim 26.

31. 27. A method of treating a disease, disorder, or condition in a subject in need of such treatment, comprising administering to the subject the pharmaceutical composition of claim 26, thereby treating the disease, disorder, or condition.

32. 32. The method of claim 31 , wherein the disease, disorder, or condition is selected from the group consisting of a rare disease, an infectious disease, a cancer, a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

33. 27. Use of the pharmaceutical composition of claim 26 in the manufacture of a medicament for the treatment of a disease, disorder, or condition.

34. 34. The use of claim 33, wherein the disease, disorder, or condition is selected from the group consisting of a rare disease, an infectious disease, a cancer, a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

35. 24. A vaccine comprising the composition of claim 23.

36. 36. The vaccine of claim 35, wherein the vaccine is selected from a tumor vaccine, an influenza vaccine, and a SARS vaccine including SARS-CoV-2.

37. 26. A vaccine comprising the concentrated lipid nanoparticle composition of claim 25.

38. 38. The vaccine of claim 37, wherein the vaccine is selected from a tumor vaccine, an influenza vaccine, and a SARS vaccine including SARS-CoV-2.

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