Drug delivery system containing oxidized cholesterol - Patents.com

Abstract

Disclosed herein is lipid nanoparticles and their compositions.Exemplary nanoparticle compositions include ionized lipid, phospholipid, PEG-lipid and cholesterol modified with hydroxyl group near D sterol ring.Disclosed nanoparticle compositions can target liver Kupffer cells and endothelial cells preferentially over hepatocytes, and should be useful for treating liver diseases whose pathogenesis involves dysfunctional Kupffer cells and endothelial cells.

Description

[Technical field]

[0001] Related Application Information This application claims priority to U.S. Patent Application No. 62 / 793,671, filed January 17, 2019, which is hereby incorporated by reference in its entirety.

[0002] Statement of Federally Funded Research This invention was made with Government support under Grants 1RO1DE026941-01A1, T32GM008433 and T32EB021962 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present invention relates generally to drug delivery systems and methods of use thereof. [Background technology]

[0004] The use of RNA to make therapeutic proteins or modify gene expression is a promising approach to treat disease. There have been some advances in local and systemic mRNA delivery (KA Hajj, KA Whitehead, Nature Reviews Materials, 2, 17056 (2017)). However, low-dose mRNA delivery to many cell types within organs and tumor microenvironments remains challenging. Identifying nanoparticles that efficiently deliver therapeutic RNA to cells within a given microenvironment is challenging because nanoparticles tend to preferentially target specific cell types, e.g., in the liver, nanoparticles preferentially target hepatocytes. Furthermore, nanoparticles that target specific cell types within a microenvironment are difficult to design, mainly due to the lack of high-throughput methods to study nanoparticle mRNA delivery in vivo.

[0005] This universal challenge in nanomedicine has slowed the development of all RNA therapeutics, as cell culture can be a poor predictor of delivery within the complex in vivo microenvironment (Paunovska, K. et al., Nano Letters, 18: 2148-2157 (2018)), forcing scientists to perform high-throughput nanoparticle assays in vitro. In vivo mRNA delivery is affected by pulsatile blood flow, heterogeneous vasculature, and clearance by the kidney, spleen, liver, lymphatics, and immune system. Barcoding technology has quantified lipid nanoparticle (LNP) biodistribution, which is necessary but not sufficient for cytoplasmic nucleic acid delivery. More specifically, less than 3% of drugs that reach target cells may escape into the cytoplasm, and the genes that alter whether nanoparticles escape into endosomes may differ for each cell type.

[0006] Therefore, there is a need for more effective nanoparticles for targeting specific cells in an organ or tumor microenvironment. Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present invention to provide compositions and methods for delivering therapeutic and prophylactic agents to the microenvironments of various organs.

[0008] It is another object of the present invention to provide nanoparticle compositions for treating and preventing diseases, including liver diseases. [Means for solving the problem]

[0009] A composition is provided for delivering nucleic acids to a specific cell or tissue microenvironment. It has been discovered that oxidized cholesterol can modify the tropism of nanoparticles. One embodiment provides nanoparticles containing oxidized cholesterol. Another embodiment provides lipid nanoparticles containing one or more oxidized cholesterols. An exemplary nanoparticle composition includes an ionized lipid, a phospholipid, one or more PEG lipids, and an oxidized cholesterol. By using a specific oxidized cholesterol, the tropism of the nanoparticles can be tailored to a desired cell type or tissue microenvironment.

[0010] Another embodiment provides nanoparticles, e.g., lipid nanoparticles containing cholesterol modified with a hydroxyl group near the D sterol ring, capable of preferentially delivering cargo to non-hepatic cells in the liver. In one embodiment, the nanoparticle composition comprises 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (CKK-E12), C 14 PEG 2000 or C 18 PEG 2000 and oxidized cholesterol modified with a hydroxyl group near the D sterol ring. The oxidized cholesterol can be 20α-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 7β,25-dihydroxylcholesterol, 27-hydroxycholesterol, 25-hydroxycholesterol, or a combination thereof.

[0011] Yet another embodiment provides a nanoparticle composition having about 30 mol% to about 80 mol% ionized lipid, about 5 mol% to about 55 mol% cholesterol, about 10 mol% to about 35 mol% phospholipid, and about 0 mol% to about 20 mol% PEG-lipid.

[0012] Another embodiment provides the disclosed nanoparticles loaded with a cargo. The cargo can be a therapeutic or prophylactic agent. In one embodiment, the therapeutic agent is a nucleic acid, including but not limited to ssRNA, dsRNA, sgRNA, mRNA, shRNA, siRNA, miRNA, ssDNA, dsDNA, antisense DNA, or combinations thereof. The nanoparticles can have a diameter of about 20 nm to about 215 nm. In one embodiment, the nanoparticles can have a diameter of about 50 nm to about 100 nm.

[0013] Another embodiment provides a pharmaceutical composition containing one or more of the disclosed lipid nanoparticles and a pharma- ceutically acceptable excipient.

[0014] Yet another embodiment provides a method for delivering a therapeutic or prophylactic agent to a subject in need thereof by administering one or more of the disclosed lipid nanoparticle compositions loaded with a therapeutic nucleic acid, such as a nucleic acid encoding a therapeutic protein, or an inhibitory or enzymatic nucleic acid. The method can further include administering a second therapeutic agent. In one embodiment, the nanoparticles preferentially deliver cargo to cells in the liver.

[0015] Another embodiment provides a method for preventing or treating liver disease in a subject by administering to the subject in need thereof one or more of the disclosed lipid nanoparticles in an amount effective for preventing or treating liver disease.The lipid nanoparticles can be administered to the subject at a dose of 0.05mg / kg.The liver disease can be liver cancer, liver cirrhosis, non-alcoholic fatty liver disease, and alcohol-related liver disease. [Brief description of the drawings]

[0016] [Figure 1-1]FIG. 1A shows various sterol variations of cholesterol. FIG. 1B shows the chemical structure of the ionization marker cKK-E12. FIG. 1C shows the chemical structure of cholesterol. FIG. 1D shows the chemical structure of C14PEG2000. FIG. 1E shows the chemical structure of C18PEG2000. FIG. 1F shows the chemical structure of DOPE. FIG. 1G is a bar graph showing the formulation ratios of ten different lipid nanoparticle designs. The graph shows the mole % of DOPE, lipid-PEG, cholesterol, and cKK-E12 in each formulation. The X-axis represents the formulation ratio and the Y-axis represents the mole % of each component of the lipid nanoparticle. [Figure 1-2] Figure 1H illustrates a workflow for barcoding the disclosed nanoparticles.Figure 1I illustrates a workflow for testing the disclosed nanoparticles. [Figure 2-1] FIG. 2A illustrates a workflow for quantifying mRNA delivery mediated by the disclosed nanoparticles. [Figure 2-2] FIG. 2B is a bar graph showing % tdTomato positive cells in 8 cell types in liver and spleen. X-axis represents cell type and Y-axis represents % tdTomato+ cells. FIG. 2C is a scatter dot plot showing the hydrodynamic diameter of all administered lipid nanoparticles. FIG. 2D is a scatter dot plot showing normalized delivery of all administered lipid nanoparticles for 8 cell types in liver and spleen. X-axis represents cell type and Y-axis represents normalized delivery. FIG. 2E shows normalized delivery for all administered lipid nanoparticles averaged across liver and spleen. [Figure 2-3] (F) Unbiased Euclidean clustering of eight different cell types from two different tissues based on lipid nanoparticle delivery cluster cell types by tissue. Asterisks represent naked barcodes. [Figure 3-1] FIG. 3A shows the mean normalized delivery of lipid nanoparticles sorted by cholesterol type. [Figure 3-2]Figure 3B shows the chemical structure of cholesterol with modifications in the cholesterol tail. Figure 3C shows the dot plots showing the normalized delivery averaged across all eight liver and spleen cell types for lipid nanoparticles containing cholesterol with modifications in the cholesterol tail (Tail) and cholesterol with modifications in the B-ring (Ring / Chol). Figure 3D shows the bar graphs showing the normalized delivery averaged across all eight liver and spleen cell types for lipid nanoparticles containing various cholesterols including 27-OH, 25-OH, 20α-OH, 7β-OH, 7β-OH, 7β-25-dihydroxy, 7-keto, 6-keto-5α-OH, and cholesterol. The X-axis represents the type of cholesterol in the lipid nanoparticles and the Y-axis represents the average whole animal normalized delivery. Figure 3E shows the enrichment of cholesterol variants in the top and bottom 10% of lipid nanoparticles. Figure 3F is a bar graph showing the enrichment factor of the top 10% of lipid nanoparticles, calculated by subtracting the enrichment of the bottom 10% of lipid nanoparticles from the enrichment of the top 10% of lipid nanoparticles. Figure 3G shows the chemical structure of ionized lipid cKK-E12. Figure 3H shows the chemical structure of C18PEG2000. Figure 3I shows the chemical structure of DOPE. Figure 3J shows the chemical structure of 20α-OH. [Figure 3-3] FIG. 3K is a diagram showing the lipid nanoparticle mole %, lipid nanoparticle diameter, polydispersity index (PDI) and pKa of each component of the two formulations. FIG. 3L is a bar graph showing the tdTomato positive endothelial cell % in mice administered 0.25 mg / kg or 50 mg / kg of LNP1 or LNP2. FIG. 3M is a bar graph showing the tdTomato positive hepatic cell % in mice administered 0.25 mg / kg or 50 mg / kg of LNP1 or LNP2. FIG. 3N is a bar graph showing the tdTomato positive Kupffer cell % in mice administered 0.25 mg / kg or 50 mg / kg of LNP1 or LNP2. FIG. 3O is a bar graph showing the tdTomato positive immune cell % in mice administered 0.25 mg / kg or 50 mg / kg of LNP1 or LNP2. The X-axis represents lipid nanoparticle dose and the Y-axis represents the tdTomato+ cell %. [Figure 4] FIG. 4A shows representative autocorrelation curves for included and excluded lipid nanoparticles. The X-axis represents time (microseconds) and the Y-axis represents autocorrelation intensity. FIG. 4B is a line graph showing diameter distribution for included and excluded lipid nanoparticles. The X-axis represents diameter (nm) and the Y-axis represents mass %. FIG. 4C is a bar graph showing % tdTomato positive cells for cell types (endothelial cells, E; immune cells, I; macrophages, M; other, O; and hematopoietic stem cells, HSC) isolated from heart, lung, kidney, pancreas, and bone marrow. The X-axis represents cell type and the Y-axis represents % tdTomato+ cells. [Figure 5-1] FIG. 5A is a chart showing enrichment (fold) difference values, mean whole animal normalized delivery (%), and enrichment calculations to obtain top and bottom 10% enrichment of lipid nanoparticles. FIG. 5B is a graph showing enrichment fold difference for cholesterol mol %. FIG. 5C is a graph showing enrichment fold difference for cholesterol mol %. FIG. 5D is a graph showing enrichment fold difference for cholesterol mol %. FIG. 5E is a graph showing enrichment fold difference for ionized lipid mol %. FIG. 5F is a graph showing enrichment fold difference for ionized lipid mol %. FIG. 5G is a graph showing enrichment fold difference for ionized lipid mol %. [Figure 5-2]FIG. 5H is a diagram showing the fold difference of enrichment for phospholipid mole %. FIG. 5I is a diagram showing the fold difference of enrichment for phospholipid mole %. FIG. 5J is a diagram showing the fold difference of enrichment for phospholipid mole %. FIG. 5K is a dot plot showing lipid nanoparticle diameter plotted against the average whole animal normalized delivery for all lipid nanoparticles. FIG. 5L is a dot plot showing lipid nanoparticle diameter plotted against the average whole animal normalized delivery for lipid nanoparticles containing only tail-modified cholesterol. FIG. 5M is a dot plot showing lipid nanoparticle diameter plotted against the average whole animal normalized delivery for lipid nanoparticles containing ring-oxidized cholesterol. FIG. 5N is a bar graph showing the % of lipid nanoparticles formed based on the cholesterol variant used. FIG. 5O is a dot plot showing lipid nanoparticle diameter based on the cholesterol variant used. [Figure 5-3] Figure 5P is a bar graph showing the % of lipid nanoparticles formed based on cholesterol mole %. Figure 5Q is a dot plot showing lipid nanoparticle diameter based on cholesterol mole %. [Figure 6-1] Figure 6A is a diagram showing CKK-E12 synthesis. Figure 6B is a diagram showing CKK-E12 synthesis. [Figure 6-2] FIG. 6C is a 1H-NMR spectrum confirming the CKK-E12 structure. [Figure 7] FIG. 7A is a bar graph showing the body weight for mice injected with 1 mg / kg lipid nanoparticle pool. FIG. 7B is a bar graph showing the body weight for mice injected with LNP1 and LNP2 containing cre mRNA at a dose of 0.25 mg / kg. FIG. 7C is a bar graph showing the body weight for mice injected with LNP1 and LNP2 containing cre mRNA at a dose of 0.05 mg / kg and 0.01 mg / kg. FIG. 7D is a bar graph showing the body weight for mice injected with LNP1 containing cre mRNA at a dose of 0.05 mg / kg compared to the control. The X-axis represents the number of days after iv injection of lipid nanoparticles, and the Y-axis represents normalized mouse body weight. PBS injection was used as a control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] I. Definition It should be understood that the present disclosure is not limited to the compositions and methods described herein, and the experimental conditions described, and therefore may vary.It should also be understood that the terms used herein are for the purpose of describing certain embodiments only, and are not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Any compositions, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.All publications mentioned are incorporated herein by reference in their entirety.

[0019] In the context of describing the invention claimed herein (particularly in the context of the claims), the use of the terms "a," "an," "the," and similar referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0020] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein.

[0021] The use of the term "about" is intended to describe values ​​above or below the stated value within approximately + / -10%; in other embodiments, values ​​may range above or below the stated value within approximately + / -5%; in other embodiments, values ​​may range above or below the stated value within approximately + / -2%; in other embodiments, values ​​may range above or below the stated value within approximately + / -1%. The ranges are intended to be made clear by the context and no further limitations are implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better clarify the invention and does not impose limitations on the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0022] As used herein, "RNA" refers to a ribonucleic acid that may or may not occur naturally. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may include a cap structure, a chain-terminating nucleoside, a stem loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence that encodes a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). The encoded polypeptide may be produced by translation of an mRNA that encodes a particular polypeptide, for example, in vivo translation of the mRNA in a mammalian cell. RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), cas9 mRNA, and mixtures thereof.

[0023] The terms "polypeptide", "peptide" and "protein" may be used interchangeably to refer to a string of at least three amino acids linked by peptide bonds. A peptide may refer to an individual peptide or a collection of peptides. A peptide may contain natural amino acids, unnatural amino acids (i.e., compounds that do not occur in nature but can be incorporated into a polypeptide chain), and / or amino acid analogs. Also, one or more of the amino acids in a peptide may be modified by addition of a chemical entity such as, for example, a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, and the like. Modifications may include cyclization of the peptide, incorporation of D-amino acids, and the like.

[0024] As used herein, "sterol" refers to organic molecules that are a subgroup of steroids. Sterols are also known as steroid alcohols. Sterols occur naturally in plants (known as phytosterols) and animals (known as zosterols).

[0025] As used herein, the terms "treat", "treating", "treatment" and "therapeutic use" refer to the elimination, reduction or amelioration of one or more symptoms of a disease or disorder. As used herein, a "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction or amelioration of such symptoms. An effect is clinically relevant if its magnitude is sufficient to affect the health or prognosis of the recipient subject. A therapeutically effective amount may refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, for example, to delay or minimize the spread of cancer. A therapeutically effective amount may also refer to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0026] As used herein, the term "prophylactic agent" refers to an agent that can be used in the prevention of a disorder or disease prior to detection of symptoms of such disorder or disease. A "prophylactically effective" amount is the amount of a prophylactic agent sufficient to mediate such protection. A prophylactically effective amount can also refer to the amount of a prophylactic agent that provides a prophylactic benefit in the prevention of disease.

[0027] As used herein, the terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans, rodents, e.g., mice and rats, and other laboratory animals.

[0028] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents.

[0029] As used herein, "Kupffer cell" refers to resident liver macrophage.Kupffer cell functions as a scavenger cell responsible for removing particulate matter from portal circulation.Kupffer cell is also involved in several pathologies of the liver, including but not limited to viral hepatitis, steatohepatitis, alcoholic liver disease, intrahepatic cholestasis, liver activation or rejection during liver transplantation, and liver fibrosis.

[0030] As used herein, "liver microenvironment" refers to the normal cells, molecules and blood vessels that surround and nourish the liver.Exemplary cells of the liver include, but are not limited to, sinusoidal endothelial cells (SECs), Kupffer cells (KCs), hepatic stellate cells (HSCs); and hepatocytes, as well as various immune cells.

[0031] "Inhibitory nucleic acid" refers to a nucleic acid that inhibits the expression of a particular gene or mRNA. Inhibitory nucleic acids include, but are not limited to, dsDNA, ssDNA, antisense DNA, siRNA, shRNA, miRNA, tasiRNA, and rasiRNA.

[0032] II. Lipid Nanoparticles Effective targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids is a continuing challenge in the field of medicine. Delivery of nucleic acids is particularly difficult due to the relative instability and low cell permeability of nucleic acids. It has been discovered that lipid nanoparticles with modified cholesterol variants can more effectively deliver nucleic acids to specific tissues in the body. In one embodiment, lipid nanoparticles can be formulated by mixing nucleic acids with ionized lipids, PEG-lipids, phospholipids, and modified cholesterol. An exemplary lipid nanoparticle formulation can include ionized lipid mimics cKK-E12, PEG-lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and one or more cholesterol variants. In one embodiment, the disclosed lipid nanoparticles preferentially target other cells (i.e., Kupffer cells, endothelial cells, immune cells) of the liver microenvironment over hepatocytes.

[0033] In one embodiment, the lipid nanoparticle formulation comprises about 30 mol% to about 80 mol% ionized lipid, about 10 mol% to about 35 mol% phospholipid, about 5 mol% to about 55 mol% modified cholesterol, and about 0 mol% to about 20 mol% PEG-lipid. In another embodiment, the lipid nanoparticle formulation comprises about 50 mol% ionized lipid, about 15 mol% phospholipid, about 30 mol% modified cholesterol, and about 5 mol% PEG-lipid. In yet another embodiment, the lipid nanoparticle formulation comprises about 30 mol% ionized lipid, about 20 mol% phospholipid, about 45 mol% modified cholesterol, and about 5 mol% PEG-lipid.

[0034] Lipid nanoparticle size can affect nanoparticle distribution. In one embodiment, lipid nanoparticles can have a diameter between about 20 and about 215 nm. Lipid nanoparticles can have a diameter that is about 25 nm, 30 nm, 35 nm, 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, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 210 nm, or 215 nm. In a preferred embodiment, the nanoparticles have a diameter between 50 nm and 100 nm.

[0035] A. Sterols The disclosed lipid nanoparticles can include one or more sterols. In one embodiment, the sterol is cholesterol. The cholesterol can be modified, e.g., oxidized. Unmodified cholesterol can be enzymatically treated to form variants that are side-chain or ring oxidized (FIG. 1A). Cholesterol can be oxidized on the beta ring structure or on the hydrocarbon tail structure. Exemplary cholesterols contemplated for use in the disclosed lipid nanoparticles include, but are not limited to, 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-25-dihydroxycholesterol, or combinations thereof. In one embodiment, side-chain oxidized cholesterol can enhance cargo delivery compared to other cholesterol variants. In a preferred embodiment, the modified cholesterol is 25-hydroxycholesterol (25-OH) or 20α-hydroxycholesterol (20α-OH).

[0036] Without being bound by any one theory, it is believed that lipid nanoparticles containing oxidized cholesterol can preferentially deliver cargo to other cells in the liver microenvironment, not just hepatocytes, as is characteristic of existing lipid nanoparticles.Cholesterol is transported in lipoproteins using forward and reverse transport, and transport to endothelial cells, hepatocytes and macrophages can be altered by cholesterol structure in vivo.In addition, lipid nanoparticles and lipoproteins have similar size and composition, therefore lipid nanoparticles with cholesterol structure incorporated therein can also be transported to cells in the same way as lipoproteins.

[0037] B. Ionized lipids In one embodiment, the disclosed lipid nanoparticles comprise ionized lipids. Ionized lipids have a positive or partial positive charge at physiological pH. Exemplary ionized lipids include, but are not limited to, 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (cKK-E12), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbanioyloxy-3-dimethylaniinopropane (DLin-C-DAP ... ,2-Dilinoleyl-3-dimethylammopropane (DLm-DAP), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-1-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)- Heptatriaeonta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (Dlin-MC3-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), dioctadecylamidoglycyocarboxysperrnine (DOGS), spermine cholesteryl carbamate (GL-67 ), bis-guanidinium-spermidine-cholesterol (BGTC), 3β-(N-(N,N'-dimethylaminoethane)carbamoyl)cholesterol (3β-(N-(N^N'-dimethylammoethanej-carbamoxlcholesterol) (DC-Chol), Nt-butyl-N'-tetradecylamino-propionamidine (diC14-amidine), dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMR1E), N,N-dioleyl-N,N-dimethyl ammonium chloride (DODAC), dioleyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DORIE), N-(1-(2,3-dioleyloxy-3)propyl-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl ammonium trifluoroacetate (DOSPA), 2-dioleoyl trimethyl ammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl ammonium chloride (DOTMA), aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE), 1,2-dioleoyl-sn-3-phosphoethanolamine ("DOPE"), or combinations thereof. In a preferred embodiment, the ionizable lipid is cKK-E12. ,

[0038] C.PEG-lipid The lipid component of the disclosed nanoparticle composition may include one or more PEG or PEG-modified lipids. Such species may alternatively be referred to as PEGylated lipids. The inclusion of PEGylated lipids may be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. PEG lipids are lipids modified with polyethylene glycol. In some embodiments, the PEGylation is reversible in that the PEG moiety is gradually released in the blood circulation. Exemplary PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DAG), PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0039] In a preferred embodiment, the PEG lipid is 14 PEG 2000or C 18 PEG 2000 It is.

[0040] D. Phospholipids The lipid component of the nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids may be associated with one or more lipid bilayers. In general, the phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0041] The phospholipid portion may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid portion may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species are also contemplated, including natural species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, the phospholipid may be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azides. Such reactions can be useful in functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane penetration or cell recognition, or in conjugating nanoparticle compositions with useful components such as targeting or imaging moieties (e.g., dyes).

[0042] Exemplary phospholipids 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 (DO ... 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (CI 6 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), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), These include distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE). In a preferred embodiment, the phospholipid is DOPE.

[0043] E. Cargo In one embodiment, the disclosed lipid nanoparticle composition comprises a therapeutic or prophylactic agent for a subject.The therapeutic or prophylactic agent can be included in the lipid nanoparticle as cargo.Exemplary cargoes that are traditionally delivered via nanoparticles include, but are not limited to, chemotherapeutic agents, cytotoxic agents, radioactive ions, small molecules, proteins, polynucleotides, and nucleic acids.

[0044] Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, lipopolysaccharide, erythropoietin ... Exemplary pro-apoptotic agents include somal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. Exemplary pro-apoptotic agents include, but are not limited to, fludarabine, staurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

[0045] Exemplary nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), RNAi inducers, RNAi agents, miRNA, antisense RNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA) ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, Cas9 / sgRNA, and plasmid DNA.

[0046] F. Pharmaceutical Compositions The nanoparticle composition can be formulated, in whole or in part, as a pharmaceutical composition. The pharmaceutical composition can include one or more nanoparticle compositions. For example, the pharmaceutical composition can include one or more nanoparticle compositions that include one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition can further include one or more pharma- ceutically acceptable excipients or accessory ingredients, such as those described herein.

[0047] Pharmaceutical compositions comprising the disclosed lipid nanoparticles are provided.The pharmaceutical compositions containing nanoparticles can be for parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (passive or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal or sublingual) administration route or using bioerodible inserts, and can be formulated into dosage forms suitable for each administration route.

[0048] In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage that is sufficient to treat, inhibit or alleviate one or more symptoms of the disorder being treated, or to provide a desired pharmacological and / or physiological effect. The exact dosage varies depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), disease, and treatment being performed.

[0049] As further studies of the disclosed nanoparticles are conducted, information will emerge regarding dosage levels suitable for the treatment of various conditions in various patients, and one of skill in the art will be able to ascertain appropriate dosing, taking into account the treatment, age, and general health of the recipient. The dosage selected will depend on the desired therapeutic effect, route of administration, and the desired duration of treatment. For the disclosed nanoparticles, dosage levels of 0.01-5 mg / kg body weight are typically administered to mammals daily. More specifically, the preferred dose for the disclosed nanoparticles is 0.05-0.25 mg / kg. Generally, for intravenous injection or infusion, dosages may be lower.

[0050] In certain embodiments, lipid nanoparticle compositions are administered locally, for example by direct injection into the site to be treated.Typically, injection results in an increased local concentration of lipid nanoparticle compositions, which is higher than can be achieved by systemic administration.The lipid nanoparticle composition can be combined with the above-mentioned matrix to help increase the local concentration of polypeptide compositions by reducing the passive diffusion of polypeptide outside the site to be treated.

[0051] 1. Formulations for parenteral administration In some embodiments, compositions disclosed herein, including those containing lipid nanoparticles, are administered in aqueous solution by parenteral injection. Formulations may also be in the form of suspensions or emulsions. In general, pharmaceutical compositions are provided that contain an effective amount of lipid nanoparticles, and these may optionally contain pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions may optionally contain one or more of the following: diluents, sterile water, buffered saline of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as surfactants and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimerosal, benzyl alcohol), and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvent or vehicle are propylene glycol, polyethylene glycol, vegetable oil, such as olive oil and corn oil, gelatin, and injectable organic ester, such as ethyl oleate.Preparation can be lyophilized and redissolved / resuspended immediately before use.Preparation can be sterilized, for example, by filtering through bacteria-retaining filter, by incorporating sterilizing agent into composition, by irradiating composition, or by heating composition.

[0052] 2. Controlled Delivery Polymer Matrices The lipid nanoparticles disclosed herein can also be administered in controlled release formulations. Controlled release polymer devices can be made for systemic long-term release after implantation or injection (microparticles) of the polymer device (rods, cylinders, films, disks). The matrix can be in the form of microparticles, such as microspheres, in which the drug is dispersed within a solid polymer matrix or microcapsules, the core being made of a material different from the polymer shell, and the peptide is dispersed or suspended in the core, which can be liquid or solid in nature. Unless otherwise defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymers can be cast as thin slabs or films ranging from nanometers to 4 centimeters, powders produced by milling or other standard techniques, or even gels, such as hydrogels.

[0053] Non-biodegradable or biodegradable matrices can be used to deliver lipid nanoparticles, but in some embodiments, biodegradable matrices are preferred. These can be natural or synthetic polymers, but in some embodiments, synthetic polymers are preferred due to their better degradation and release profile characteristics. The polymer is selected based on the period of time over which release is desired. In some cases, linear release may be most useful, while in other cases, pulsed or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% water by weight) and may be optionally crosslinked with multivalent ions or polymers.

[0054] The matrix can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed to prepare microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0055] The devices can be formulated for local release to treat the area of ​​implantation or injection - typically delivering a much smaller dose than would be required to treat the entire body - or for systemic delivery. They can be implanted or injected subcutaneously, into muscle, into fat, or swallowed.

[0056] III.How to use Disclosed herein are methods of using the disclosed lipid nanoparticles to deliver cargo to specific cells or organs.In some embodiments, the nanoparticles can deliver therapeutic or prophylactic agents to specific cells or organs of a subject in need thereof.In another embodiment, the disclosed lipid nanoparticles can be used to treat or prevent diseases in a subject in need thereof.

[0057] A. Methods for Delivering Cargo to Cells and Organs Provided herein are methods for delivering therapeutic and / or prophylactic agents to a subject in need thereof.

[0058] In some embodiments, the disclosed lipid nanoparticle compositions can target a specific type or class of cells (e.g., cells of a particular organ or system). For example, a nanoparticle composition containing a therapeutic and / or prophylactic agent of interest can be specifically delivered to the liver, kidney, spleen, intestine, or lungs of a mammal. Specific delivery to a particular class of cells, organ, or system, or group thereof, means that a nanoparticle composition containing a higher percentage of the therapeutic and / or prophylactic agent is delivered to the desired destination (e.g., tissue) compared to other destinations. In some embodiments, specific delivery can result in a greater than 2-fold, greater than 5-fold, greater than 10-fold, greater than 15-fold, or greater than 20-fold increase in the amount of therapeutic and / or prophylactic agent per gram of tissue of the targeted destination.

[0059] Another embodiment provides targeted or specific delivery of mRNA encoding a protein binding partner such as an antibody or functional fragment thereof, a scaffold protein, or a peptide, or a receptor on the cell surface. The mRNA may additionally or alternatively be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biomolecules. Alternatively, other therapeutic and / or prophylactic agents, or elements of the nanoparticle composition, may be selected based on their affinity for a particular receptor, so that the nanoparticle composition can more rapidly interact with a target cell population that contains the receptor. For example, the ligand may include, but is not limited to, members of a specific binding pair, antibodies, monoclonal antibodies, Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof; multivalent binding reagents including mono- or bispecific antibodies, such as disulfide-stabilized Fv fragments, scFv tandems, diabodies, tribodies, or tetrabodies; as well as aptamers, receptors, and fusion proteins.

[0060] Targeted cells may include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac myocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0061] In one embodiment, the disclosed lipid nanoparticles target cells of the liver microenvironment, excluding hepatocytes. These cells can be liver endothelial cells, Kupffer cells, and immune cells.

[0062] In another embodiment, the disclosed lipid nanoparticles target cells of the immune system.Exemplary immune cells include, but are not limited to, granulocytes, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, including cytotoxic T cells, T helper cells, and regulatory T cells.

[0063] B. Methods of Modulating the Immune Response The disclosed lipid nanoparticles can be used to regulate the immune response of subjects who need such treatment.In one embodiment, the disclosed lipid nanoparticles target immune cells of diseased organs, organ system microenvironments, or tumors.The immune cells can be macrophages, more specifically Kupffer cells.

[0064] 1. Stimulating immune response Treatment strategy A method for inducing or enhancing immune response in a subject is provided.Typically, the method comprises administering to a subject an effective amount of the disclosed lipid nanoparticles or a pharmaceutical composition comprising the disclosed lipid nanoparticles.The immune response can be, for example, the stimulation of innate immunity at disease or tumor sites, more specifically the stimulation of Kupffer cells.Kupffer cells are the first macrophage population of the body that comes into contact with bacteria, bacterial endotoxins and microbial debris derived from the digestive tract and transported to the liver via the portal vein.

[0065] Alternatively, lipid nanoparticles can stimulate signaling through immune cell receptors to promote or enhance the immune response.

[0066] In some embodiments, lipid nanoparticles can be used to block the suppressive immune cells to tumor microenvironment.In another embodiment, lipid nanoparticles can be used to inhibit, reduce or block tumor metastasis.In some embodiments, lipid nanoparticles can reduce or inhibit the activity of immunosuppressive macrophages, reduce the production of cytokines such as IL-12 by M1 macrophages, reduce macrophage differentiation, reduce the number of macrophages, reduce the ratio of macrophages in immune cell populations, or reduce macrophage survival.

[0067] The method can be used in vivo or ex vivo to inhibit, reduce, or block suppressive immune responses, thereby having a stimulating therapeutic effect. In some embodiments, lipid nanoparticles are administered directly to a subject. In some embodiments, lipid nanoparticles are contacted with cells (e.g., immune cells) ex vivo, and the treated cells are administered to a subject (e.g., adoptive transfer). Lipid nanoparticles can enable a more robust immune response. The disclosed compositions are useful for stimulating or enhancing immune responses involving T cells by inhibiting, reducing, or blocking suppressive immune signaling through T cell receptors.

[0068] b. Subject to treatment i. Cancer Treatment The disclosed compositions and methods can be used to treat cancer.Generally, lipid nanoparticles are used to stimulate or enhance the immune response of a subject against cancer by administering a certain amount of lipid nanoparticles to the subject.The method can reduce one or more symptoms of cancer.In one embodiment, the disclosed lipid nanoparticles inhibit, block or reduce the number of macrophages at tumor site, thereby reducing tumor angiogenesis and the suppression of anti-tumor immune cells.

[0069] Cancer cells acquire a characteristic set of functional capabilities during their development through various mechanisms. Such capabilities include evasion of apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, limitless replicative potential, and sustained angiogenesis. The term "cancer cells" is intended to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to benign tumors that remain localized. In other embodiments, cancer refers to malignant tumors that have invaded and destroyed adjacent body structures and spread to distant sites. In still other embodiments, cancer is associated with a specific cancer antigen (e.g., pan-cancer antigen (KS1 / 4), ovarian cancer antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[0070] The methods and compositions disclosed herein are useful for the treatment or prevention of various cancers or other abnormal proliferative disorders, including, but not limited to, cancers of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; cancers, including squamous cell carcinoma; hematopoietic malignancies of the lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; acute and chronic myeloid leukemia. hematopoietic tumors of myeloid lineage, including idiopathic leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratocarcinoma.

[0071] Cancer caused by abnormality of apoptosis can also be treated by the disclosed method and composition. Such cancers can include, but are not limited to, follicular lymphoma, cancer with p53 mutation, hormone-dependent tumors of breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndrome. In a specific embodiment, malignant tumor or dysproliferative change (such as metaplasia and dysplasia), or hyperproliferative disorder is treated or prevented by the method and composition in ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented by the method and composition.

[0072] Specific cancers and related disorders that can be treated or prevented by the methods and compositions disclosed herein include, but are not limited to, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemic leukemia and myelodysplastic syndromes, chronic leukemias, such as but not limited to chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as but not limited to Hodgkin's disease or non-Hodgkin's disease lymphomas (e.g. for example, diffuse anaplastic lymphoma kinase (ALK) negative, large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (ALK) positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK) positive, ALK+ anaplastic large cell lymphoma (ALCL), acute myeloid lymphoma (AML); multiple myeloma, for example, but not limited to, smoldering multiple myeloma, non-secretory myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom macroglobulinemia, monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; sarcomas of bone and connective tissue, including, but not limited to, sarcoma of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; including, but not limited to, glioma, astrocytoma, brain stem nerve brain tumors, including glioma, ependymoma, oligodendroglioma, non-glial tumors, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary carcinoma of the breast, mucinous carcinoma of the breast, tubular carcinoma of the breast, papillary breast carcinoma, Paget's disease and inflammatory breast cancer; adrenal gland cancer, including but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer, for example, but not limited to, papillary or follicular thyroid cancer, medullary thyroid carcinoma and anaplastic thyroid carcinoma;pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumors, and carcinoid or islet cell tumors; pituitary cancer, including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancer, including but not limited to intraocular melanoma, e.g., iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancer, including but not limited to ovarian epithelial carcinoma, borderline tumor, germ cell tumor and stromal tumor; esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma and oat cell (small cell) carcinoma; adenocarcinoma, including but not limited to adenocarcinoma, fungiform (polypoid), ulcerating, superficial spreading, diffuse spreading, malignant lymphoma, fatty meat gastric cancer, including but not limited to sarcoma, fibrosarcoma and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancer, including but not limited to adenocarcinoma; cholangiocarcinoma, including but not limited to papillary, nodular and diffuse; lung cancer, including but not limited to non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer; testicular cancer, including but not limited to germinal tumors, seminoma, undifferentiated, classical (typical), spermatocyte, non-seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma (yolk sac tumor), prostate cancer, including adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer; oral cancer, including but not limited to squamous cell carcinoma; basal carcinoma; salivary gland cancer, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to squamous cell carcinoma and verrucous carcinoma; skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentigo melanoma; kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter);Wilms' tumor; bladder cancer, including, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteogenic sarcoma, endothelial cell sarcoma, lymphangioendothelial cell sarcoma, mesothelioma, synovium, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (for a summary of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America);

[0073] ii. Treatment of Infectious Diseases The disclosed lipid nanoparticles can be used to treat infectious diseases and infectious diseases. In general, lipid nanoparticles are used to stimulate or enhance the immune response of a subject to an infectious disease. The disclosed lipid nanoparticles can stimulate macrophages, more specifically, Kupffer cells in the liver. In one embodiment, the lipid nanoparticles inhibit, reduce, or block suppressive immune signaling. In another embodiment, the lipid nanoparticles induce, promote, or enhance immune response by inducing, promoting, or enhancing signaling through immune cell receptors. The method can reduce one or more symptoms of an infectious disease.

[0074] Infection or disease can be caused by bacteria, viruses, protozoa, helminths or other microbial pathogens that invade cells and are thus attacked by cytotoxic T lymphocytes.

[0075] Infection or disease can be acute or chronic. Acute infection is typically short-lasting infection. During acute microbial infection, immune cells begin to express immunomodulatory receptors. Thus, in some embodiments, the method comprises increasing the immune stimulatory response to acute infection.

[0076] The infection may be caused by, for example, but not limited to, Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacterium.

[0077] In some embodiments, the disclosed compositions are used to treat chronic infections, such as infections in which T cell depletion or T cell anergy occurs, allowing the infection to persist in the host for extended periods of time.

[0078] Exemplary infections to be treated are chronic infections caused by hepatitis virus, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes virus, Epstein-Barr virus, or human papilloma virus.

[0079] Because bacterial infections are primarily cleared by macrophages, increasing macrophage activity would be therapeutically useful in situations where more rapid or complete clearance of infectious bacterial agents would be beneficial to an animal or human subject. Thus, the disclosed compositions can be administered to treat local or systemic bacterial infections.

[0080] Representative infections that can be treated include, but are not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, and the like. ), Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type BB) (HIB), Hyphomicrobium, Legionella, Leptospirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas udomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus and Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatisThese include infections caused by microorganisms including Plasmodium trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni.

[0081] Other microorganisms that can be treated using the disclosed compositions and methods include bacteria, such as those of the genera Klebsiella, Serratia, Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or fungal or parasitic pathogens, such as Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus, Aspergillus (fumigatus, niger, etc.), and the like. (niger, etc.), genera of the order Mucorales (mucor, absidia, rhizophus), the genera Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis) and the genera Histoplasma (capsulatuma), Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba spp., Giardia lambia, Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondiigondi, etc.), Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia, or Trypanosoma.

[0082] 2. Immune response inhibition Treatment strategy A method is provided for reducing or inhibiting the immune response of a subject.Typically, the method comprises administering to a subject an effective amount of the disclosed lipid nanoparticles or cells primed ex vivo by these nanoparticles.The immune response can be, for example, promoting or enhancing an inhibitory immune response.In one embodiment, the disclosed composition reduces, blocks or reduces the activity of M1 macrophages or M1 macrophage signaling, and reduces the production of cytokines such as IL-12, resulting in an immunosuppressive response.

[0083] In another embodiment, the lipid nanoparticles promote an inhibitory immune response by inducing, promoting or enhancing signaling through immune cell receptors.

[0084] The method can be used as an immune response inhibition therapeutic application in vivo or ex vivo. Thus, in some embodiments, lipid nanoparticles are directly administered to a subject. In some embodiments, lipid nanoparticles are contacted with cells (e.g., immune cells) ex vivo, and the treated cells are administered to a subject (e.g., adoptive transfer). In general, the disclosed lipid nanoparticles can be used to treat a subject having or susceptible to any disease or disorder in which the subject's immune system initiates an overactive or inappropriate immune response. The agent can allow for a less robust immune response. The disclosed compositions are useful for reducing or inhibiting immune responses involving T cells.

[0085] B inflammatory response The disclosed lipid nanoparticles can be used to treat inflammation.Generally, a drug is used to reduce or inhibit the immune response of a subject by administering a certain amount of the disclosed lipid nanoparticles to the subject.The method can reduce one or more symptoms of inflammation.The inflammation can be acute, chronic or persistent inflammation.

[0086] In some embodiments, lipid nanoparticles slow down immune system.For example, the drug can be used to control the excessive inflammatory response that causes damage to healthy tissue.Therefore, in some embodiments, the drug is administered to the subject experiencing the excessive inflammatory response.In such cases, it may be beneficial for the subject to control the excessive immune response.

[0087] c. Inflammatory and autoimmune diseases / disorders The disclosed lipid nanoparticles can also be used to treat inflammatory or autoimmune diseases and disorders. Representative inflammatory or autoimmune diseases / disorders include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue syndrome-immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyalgia thyroiditis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus (type I), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0088] In some embodiments, the inflammation or autoimmune disease is caused by a pathogen or is the result of an infection.

[0089] C. Methods of Treating Liver Disease The disclosed methods and compositions can be used to reduce, decrease, prevent, or limit the onset, development, progression, signs or symptoms of liver disease and disorders.Dysfunctioning liver endothelial cells, Kupffer cells, and other cells in the liver microenvironment cause disease rather than hepatocytes.Historically, nanoparticles tend to preferentially target hepatocytes rather than other cells in the liver microenvironment.

[0090] Without being bound by any one theory, it is believed that the cholesterol modifications disclosed herein enable lipid nanoparticles to more potently deliver cargo to cells within the liver microenvironment in vivo.

[0091] In one embodiment, the disclosed nanoparticles can be used for protein replacement therapy within the liver microenvironment.

[0092] The lipid nanoparticles can be administered locally or systemically in an amount effective to induce a local or systemic response, for example, to treat or prevent liver disease. In a preferred embodiment, one or more lipid nanoparticles are administered once per week at a dose of 0.05-0.25 mg / kg body weight. In another embodiment, the nanoparticles are delivered twice per month, once per month, or less frequently. Exemplary liver diseases and disorders that can be treated by lipid nanoparticles are provided below.

[0093] 1. Liver cancer The disclosed methods and compositions can be used to reduce, reduce, prevent, or limit the onset, development, progression, signs or symptoms of liver cancer.In some embodiments, the liver cancer treated is asymptomatic liver cancer.In some embodiments, liver cancer is identified by detecting diagnostic markers related to the onset, development or progression of liver cancer.In further embodiments, risk factors for the development of liver cancer are used as a mechanism to identify subjects who can benefit from preventive treatment with the disclosed methods and compositions.

[0094] In one embodiment, the disclosed lipid nanoparticles can deliver chemotherapeutic agents to the liver to treat liver cancer and / or reduce tumor burden. In another embodiment, the disclosed lipid nanoparticles can deliver apoptotic agents to the liver to treat liver cancer and / or reduce tumor burden.

[0095] 2. Liver cirrhosis The disclosed methods and compositions can be used to reduce, decrease, prevent, or limit the onset, development, progression, signs or symptoms of cirrhosis.

[0096] Cirrhosis is a disease in which liver cells are damaged and replaced by scar tissue. Factors that contribute to cirrhosis include, but are not limited to, infectious diseases, alcohol abuse, recreational drug abuse, and non-fatty liver disease. Cirrhosis is associated with the development of liver cancer. Chronic infection with Hepatitis C Virus (HCV) has been recognized as an increased risk for hepatocellular carcinoma (HCC). Approximately 20% of HCV-infected individuals have disease that progresses to cirrhosis, and about 40% of these patients develop HCC after an average of 10-15 years. Cirrhosis of the liver can also be caused by infection with Hepatitis B Virus (HBV).

[0097] In one embodiment, the disclosed lipid nanoparticles can deliver therapeutic agents to the liver to treat symptoms of cirrhosis or halt the progression of the disease.

[0098] 3. Fatty liver disease The disclosed methods and compositions can be used to reduce, decrease, prevent, or limit the onset, development, progression, signs or symptoms of fatty liver disease. Fatty liver disease can be a result of alcohol abuse, which is the leading cause of cirrhosis in the United States.

[0099] Fatty liver disease can also be non-alcoholic fatty liver disease. Cirrhosis of the liver can be caused by non-alcoholic fatty liver disease, a condition in which people who consume little or no alcohol develop fatty liver. Non-alcoholic fatty liver disease is common in obese people. People with a form of this disease known as non-alcoholic steatohepatitis (NASH) can progress to the development of cirrhosis. Type 2 diabetes has been associated with an increased risk of liver cancer, especially in patients who also have other risk factors such as heavy alcohol use and / or chronic viral hepatitis. This risk can be increased because people with type 2 diabetes tend to be overweight or obese, which in turn can cause liver problems.

[0100] In some embodiments, the disclosed lipid nanoparticles are used prophylactically.Thus, lipid nanoparticles can be administered daily in the absence of symptoms or markers of liver disease to promote overall liver health and prevent the onset of liver disease in individuals at risk of liver disease.

[0101] IV. Combination Therapy The disclosed lipid nanoparticles can be administered to a subject in need thereof alone or in combination with one or more additional therapeutic agents.In some embodiments, lipid nanoparticles and additional therapeutic agents are administered separately but simultaneously.Lipid nanoparticles and additional therapeutic agents can also be administered as part of the same composition.In other embodiments, lipid nanoparticles and second therapeutic agents are administered separately and at different times but as part of the same treatment regime.

[0102] The subject may be administered the first therapeutic agent 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours or more, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more, prior to administration of the second therapeutic agent. In some embodiments, the subject may be administered one or more doses of the first agent every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 21 days, 28 days, 35 days or 48 days prior to the first administration of the second agent. The lipid nanoparticle may be the first or second therapeutic agent.

[0103] Lipid nanoparticles and additional therapeutic agents can be administered as part of a treatment regimen.For example, a first therapeutic agent can be administered to a subject every 4 days, and a second therapeutic agent can be administered on the first, second, third or fourth day, or a combination thereof.The first therapeutic agent or the second therapeutic agent can be administered repeatedly throughout the treatment regimen.

[0104] Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutic agents, enzymes, antibiotics, antivirals (particularly protease inhibitors alone or in combination with nucleosides to treat HIV or Hepatitis B or C), antiparasitic agents (helminths, protozoans), growth factors, growth inhibitory agents, hormones, hormone antagonists, antibodies and biologically active fragments thereof (including humanized, single chain, and chimeric antibodies), antigens and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatory drugs, ligands that bind to Toll-like receptors to activate the innate immune system (including, but not limited to, CpG oligonucleotides), molecules that mobilize and optimize the adaptive immune system, and other molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells and helper T cells, as well as other molecules that deactivate or downregulate suppressor or regulatory T cells.

[0105] The additional therapeutic agent is selected based on the condition, disorder or disease to be treated. For example, the immunomodulatory agent can be co-administered with one or more additional agents that function to enhance or promote immune response, or to reduce or inhibit immune response.

[0106] A. Increasing the immune response 1. Antimicrobial drugs For example, the disclosed lipid nanoparticles can be used in a prophylactic or preventative role in the treatment and prevention of the diseases discussed above, as well as in the context of severe traumatic injuries, such as large burns, open fractures, accidental amputations or other wounds. Thus, the disclosed lipid nanoparticles can be administered to a subject in combination with antimicrobial agents, such as antibiotics, antifungals, antivirals, antiparasitics, or essential oils.

[0107] In some embodiments, subjects are administered lipid nanoparticles and / or antimicrobial drugs upon admission to prevent further bacterial, fungal or viral complications. Antibiotics can target pathogens and lipid nanoparticles can stimulate the immune system to provide an enhanced response to treat or prevent further infection or disease.

[0108] 2. Chemotherapeutic agents The disclosed lipid nanoparticles can be combined with one or more chemotherapeutic and pro-apoptotic agents. Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, lipopolysaccharide, erythropoietin ... Exemplary pro-apoptotic agents include somal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. Exemplary pro-apoptotic agents include, but are not limited to, fludarabine, staurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

[0109] 3. Other immunomodulatory drugs a.PD-1 antagonist In some embodiments, the lipid nanoparticles are co-administered with a PD-1 antagonist. Programmed cell death-1 (PD-1) is a member of the CD28 family of receptors that, when induced on T cells, results in a negative immune response. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell proliferation and / or the strength and / or duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Pat. Nos. 8,114,845, 8,609,089 and 8,709,416, the entireties of which are specifically incorporated herein by reference, and include compounds or agents that bind to and block the ligand of PD-1, preventing or inhibiting the binding of the ligand to the PD-1 receptor, or that directly bind to and block the PD-1 receptor without inducing inhibitory signaling through the PD-1 receptor.

[0110] In some embodiments, a PD-1 receptor antagonist binds directly to the PD-1 receptor without triggering inhibitory signaling, and also binds to a ligand of the PD-1 receptor, reducing or preventing the ligand from triggering signaling through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and trigger the transmission of inhibitory signals, fewer cells are attenuated by the negative signals delivered by PD-1 signaling, and a more robust immune response can be achieved.

[0111] PD-1 signaling is thought to be driven by binding of PD-1 ligands (such as B7-H1 or B7-DC) in close proximity to peptide antigens presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Thus, proteins, antibodies, or small molecules that prevent co-ligation of PD-1 and the TCR on the T cell membrane are also useful PD-1 antagonists.

[0112] In some embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or prevents PD-1 receptor signaling by binding to a ligand of PD-1 or to PD-1 itself, particularly in cases where co-ligation of PD-1 and the TCR is not amenable to binding and thus does not trigger inhibitory signaling through the PD-1 receptor.

[0113] Other PD-1 antagonists contemplated by the methods of the invention include antibodies that bind to PD-1 or ligands of PD-1, as well as other antibodies.

[0114] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following U.S. Patents: 7,332,582, 7,488,802, 7,521,051, 7,524,498, 7,563,869, 7,981,416, 8,088,905, 8,287,856, 8,5 These include those described in Nos. 80247, 8728474, 8779105, 9067999, 9073994, 9084776, 9205148, 9358289, 9387247, 9492539, and 9492540.

[0115] See also Berger et al., Clin. Cancer Res., 14:3044-3051 (2008).

[0116] Exemplary anti-B7-H1 (also referred to as anti-PD-L1) antibodies include, but are not limited to, those described in the following U.S. Patent Nos. 8,383,796, 9,102,725, 9,273,135, 9,393,301, and 9,580,507, all of which are specifically incorporated by reference herein in their entireties.

[0117] For anti-B7-DC (also known as anti-PD-L2) antibodies, see U.S. Patent Nos. 7,411,051, 7,052,694, 7,390,888, 8,188,238, and 9,255,147, all of which are specifically incorporated by reference in their entireties.

[0118] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologs and variants thereof, and active fragments of any of the foregoing, as well as fusion proteins incorporating any of the above. In some embodiments, the fusion protein comprises a soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or a portion of the transmembrane portion of human B7-DC.

[0119] The PD-1 antagonist can also be a fragment of mammalian B7-H1, e.g., from a mouse or a primate, e.g., a human, that binds to and blocks PD-1 but does not result in inhibitory signaling through PD-1. The fragment can also be part of a fusion protein, e.g., an Ig fusion protein.

[0120] Other useful polypeptide PD-1 antagonists include those that bind to the ligand of the PD-1 receptor. These include PD-1 receptor proteins, or soluble fragments thereof, that can bind to the PD-1 ligand, such as B7-H1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signaling. B7-H1 has also been shown to bind to the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include soluble ECD portions of the PD-1 protein that contain mutations, such as the A99L mutation, that increase binding to the natural ligand (Molnar et al., PNAS, 105:10483-10488 (2008)). Also useful are B7-1 or soluble fragments thereof that can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signaling.

[0121] PD-1 and B7-H1 antisense nucleic acids (both DNA and RNA), as well as siRNA molecules, can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells and the production of T cell ligands, such as B7-H1, PD-L1 and / or PD-L2. For example, siRNA (e.g., about 21 nucleotides long, specific to the gene encoding PD-1 or encoding PD-1 ligand, and the oligonucleotides can be easily purchased commercially) complexed with a carrier such as polyethylenimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8): 2231-2244 (2009)) can be easily taken up by cells expressing PD-1 and PD-1 ligands, reducing the expression of these receptors and ligands, achieving a reduction in inhibitory signaling in T cells, thereby activating T cells.

[0122] b. CTLA4 antagonist Other molecules useful for mediating the effect of T cells in immune response are also contemplated as additional therapeutic agents.In some embodiments, the molecule is an antagonist of CTLA4, such as an antagonist anti-CTLA4 antibody.Examples of anti-CTLA4 antibodies contemplated for use in the methods of the present invention include the antibodies described in PCT / US2006 / 043690 (Fischkoff et al., International Publication WO 2007 / 056539).

[0123] Dosages for anti-PD-1, anti-B7-H1 and anti-CTLA4 antibodies are known in the art and may range, for example, from 0.1 to 100 mg / kg, or shorter ranges of 1 to 50 mg / kg or 10 to 20 mg / kg. Suitable doses for human subjects may be between 5 and 15 mg / kg, with 10 mg / kg of antibody (e.g., human anti-PD-1 antibody) being a specific embodiment.

[0124] Specific examples of anti-CTLA4 antibodies useful in the methods of the invention are the human anti-CTLA4 antibody ipilimumab, administered at a dose of, e.g., about 10 mg / kg, and the human anti-CTLA4 antibody tremelimumab, administered at a dose of, e.g., about 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[0125] In another embodiment, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to B7-1 ligand and prevent it from binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002)). These small organic compounds can be administered alone or together with anti-CTLA4 antibody to reduce the inhibitory signaling of T cells.

[0126] 4. Enhancers In some embodiments, the additional therapeutic agent comprises a potentiator, which acts to increase the effectiveness of the immune response upregulator, possibly by more than one mechanism, although the precise mechanism of action is not essential to the broad practice of the invention.

[0127] In some embodiments, the potentiator is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan® or Neosar®) is an oxazahosphorine drug, and analogs include ifosfamide (IFO, Ifex), perphosfamide, trophosfamide (trophosfamide; Ixoten), and pharma- ceutically acceptable salts, solvates, prodrugs, and metabolites thereof (US Patent Publication No. 20070202077, incorporated in its entirety). Ifosfamide (MITOXANA®) is a structural analog of cyclophosphamide, and its mechanism of action is believed to be the same as or substantially similar to that of cyclophosphamide. Perphosfamide (4-hydroperoxycyclophosphamide) and trophosfamide are also alkylating agents structurally related to cyclophosphamide. For example, perphosfamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. In an attempt to improve selectivity and response with reduced host toxicity, new oxazaphosphorine derivatives have been designed and evaluated (Liang J, Huang M, Duan W, Yu XQ, Zhou S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des. 2007;13(9):963-78. Review). These include mafosfamide (NSC345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide mustard), S-(-)-bromophosphamide (CBM-11), NSC612567 (aldofosphamide perhydrothiazine) and NSC613060 (aldofosphamide thiazolidine). Mahofosfamide is an oxazaphosphorine analogue that is a chemically stable 4-thioethanesulfonate salt of 4-hydroxy-CPA. Glufosfamide is an IFO derivative in which an alkylated metabolite of IFO, isophosphoramide mustard, is glycosidically linked to a beta-D-glucose molecule.Additional cyclophosphamide analogs are described in US Pat. No. 5,190,929, entitled "Cyclophosphamide Analogs Useful as Antitumor Agents," which is incorporated herein by reference in its entirety.

[0128] Although CTX itself is non-toxic, some of its metabolites are cytotoxic alkylating agents that induce DNA cross-links and, at high doses, strand breaks. Many cells are resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes, since lymphocytes (except hematopoietic stem cells) express only low levels of ALDH, and cycling cells are most sensitive to DNA alkylating agents.

[0129] Low doses of CTX (less than 200 mg / kg) can have immune stimulatory effects, including stimulation of antitumor immune responses in humans and mouse models of cancer (Brode & Cooke Crit Rev. Immunol. 28:109-126 (2008)). These low doses are subtherapeutic and have no direct antitumor activity. In contrast, high doses of CTX inhibit antitumor responses. Several mechanisms may explain the role of CTX in enhancing antitumor immune responses: (a) depletion of CD4+CD25+FoxP3+Tregs (and specifically proliferating Tregs, which may be particularly suppressive), (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), which leads to suppression of tumor cell proliferation; (d) recruitment and expansion of CD11b+Gr-1+MDSCs. These primary effects have numerous secondary effects; for example, after Treg depletion, macrophages produce more IFN-γ and less IL-10. CTX has also been shown to induce type I IFN expression and promote homeostatic proliferation of lymphocytes.

[0130] Treg depletion is the most commonly cited mechanism by which CTX enhances antitumor immune responses. This conclusion is based in part on the results of adoptive transfer experiments. In the AB1-HA tumor model, CTX treatment on day 9 results in a 75% cure rate. Transfer of purified Tregs on day 12 almost completely inhibited the CTX response (van der Most et al. Cancer Immunol. Immunother. 58:1219-1228 (2009)). Similar results were observed in the HHD2 tumor model: adoptive transfer of CD4+CD25+Tregs after CTX pretreatment eliminated the therapeutic response to the vaccine (Taieb, JJ Immunol. 176:2722-2729 (2006)).

[0131] Numerous human clinical trials have demonstrated that low-dose CTX is a safe, well-tolerated, and effective agent for promoting anti-tumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47:1-12 (1998)).

[0132] The optimal dose of CTX to enhance anti-tumor immune responses is one that reduces total T cell numbers by reducing Treg levels below the normal range, but is sub-therapeutic (see Machiels et al. Cancer Res. 61:3689-3697 (2001)).

[0133] In human clinical trials where CTX has been used as an immune enhancer, a dose of 300 mg / m2 has typically been used. For an average male (6 feet, 170 pounds (78 kg) with a body surface area of ​​1.98 m2), 300 mg / m2 is 8 mg / kg, or 624 mg of total protein. In mouse models of cancer, efficacy has been seen at doses ranging from 15 to 150 mg / kg, associated with 0.45 to 4.5 mg of total protein in a 30 g mouse (Machiels et al. Cancer Res. 61:3689-3697 (2001); Hengst et al Cancer Res. 41:2163-2167 (1981); Hengst Cancer Res. 40:2135-2141 (1980)).

[0134] In large mammals, such as primates, such as humans, such mg / m2 doses can be used, but also unit doses administered over a finite time interval can be used.Such unit doses can be administered daily over a finite period, for example, up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days are all specifically contemplated by the present invention.The same regimen can also be applied to other augmenting agents listed herein.

[0135] In other embodiments, the potentiating agent is an agent that reduces the activity and / or number of regulatory T lymphocytes (T-reg), such as sunitinib (SUTENT®), anti-TGFβ, or imatinib (GLEEVAC®). The listed treatment regimes may also include administering an adjuvant.

[0136] Useful enhancing agents also include antimitotic agents such as paclitaxol, aromatase inhibitors (e.g., letrozole) and angiogenesis inhibitors (VEGF inhibitors, e.g., Avastin, VEGF-Trap) (see, e.g., Li et al., Vascular endothelial growth factor blockade reduces intratumoral regulatory T cells and enhances the efficacy of a GM-CSF-secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov 15; 12(22):6808-16.), anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18 antagonists.

[0137] B. Reduced immune response 1. Immunosuppressants In some embodiments, an immune response or an inflammatory / autoimmune disease / disorder is treated by administering to a subject the disclosed lipid nanoparticles and a second agent that is an immunosuppressant. Immunosuppressants include, but are not limited to, antibodies against other lymphocyte surface markers (e.g., CD40, alpha-4 integrin) or cytokines, fusion proteins (e.g., CTLA-4-Ig (Orencia®), TNFR-Ig (Enbrel®)), TNF-α blockers such as Enbrel, Remicade, Cimzia, and Humira, cyclophosphamide (CTX) (i.e., Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), methotrexate (MTX) (i.e., Rheumatrex®, Trexall®), belimumab (i.e., Benlysta®), or other immunosuppressants (e.g., cyclosporine A, FK506-like compounds, rapamycin compounds, or steroids), antiproliferative agents, cytotoxic agents, or other compounds that may aid in immunosuppression.

[0138] The therapeutic agent may be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). The CTLA-4-Ig fusion protein functions to compete with the costimulatory receptor, CD28, on T cells for binding to CD80 / CD86 (B7-1 / B7-2) on antigen presenting cells, thus inhibiting T cell activation. In another embodiment, the therapeutic agent is a CTLA-4-Ig fusion protein known as belatacept. Belatacept contains two amino acid substitutions (L104E and A29Y) that significantly increase its binding activity to CD86 in vivo. In another embodiment, the therapeutic agent is Maxy-4.

[0139] In another embodiment, the therapeutic agent is cyclophosphamide (CTX). Cyclophosphamide, also known as cyclophosphan (generic name for Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), is a nitrogen mustard alkylating agent of the oxazophorine group. It is used to treat various types of cancer and some autoimmune disorders. Cyclophosphamide (CTX) is the primary drug used for diffuse proliferative glomerulonephritis in patients with renal lupus.

[0140] Therapeutic agents can be administered in an amount effective to reduce blood or serum levels of anti-double stranded DNA (anti-dsDNA) autoantibodies and / or reduce proteinuria in a patient in need thereof.

[0141] In another embodiment, the therapeutic agent increases the amount of adenosine in serum.See, for example, WO 08 / 147482.For example, the second therapeutic agent can be CD73-Ig, recombinant CD73, or another agent (e.g., cytokine or monoclonal antibody or small molecule) that increases the expression of CD73.See, for example, WO 04 / 084933.In another embodiment, the therapeutic agent is interferon-beta.

[0142] The Therapeutic Agent may be a small molecule that inhibits or reduces differentiation, proliferation, activity and / or cytokine production and / or secretion by Th1, Th17, Th22, and / or other cells that secrete or cause other cells to secrete inflammatory molecules including, but not limited to, IL-1β, TNF-α, TGF-beta, IFN-γ, IL-18, IL-17, IL-6, IL-23, Il-22, IL-21, and MMPs. In another embodiment, the Therapeutic Agent is a small molecule that interacts with Tregs, enhances Treg activity, promotes or enhances IL-10 secretion by Tregs, increases the number of Tregs, increases the suppressive capacity of Tregs, or a combination thereof.

[0143] In some embodiments, the composition increases Treg activity or production. Exemplary Treg enhancers include, but are not limited to, glucocorticoid fluticasone, salmeterol, IL-12, IFN-γ and antibodies against IL-4; vitamin D3 and dexamethasone, and combinations thereof.

[0144] In some embodiments, the therapeutic agent is an antibody, for example a function-blocking antibody against a pro-inflammatory molecule such as IL-6, IL-23, IL-22 or IL-21.

[0145] As used herein, the term "rapamycin compounds" includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds believed to have the same mechanism of action as rapamycin (e.g., inhibition of cytokine function). The term "rapamycin compounds" includes compounds that have structural similarity to rapamycin, e.g., compounds with similar macrocyclic structures that have been modified to enhance their therapeutic effectiveness. Exemplary rapamycin compounds are known in the art (see, e.g., WO 95122972, WO 95116691, WO 95104738, U.S. Pat. Nos. 6,015,809; 5,989,591; 5,567,709; 5,559,112; 5,530,006; 5,484,790; 5,385,908; 5,202,332; 5,162,333; 5,780,462; 5,120,727).

[0146] The term "FK506-like compounds" includes FK506, as well as FK506 derivatives and analogs, such as compounds with structural similarity to FK506, such as compounds with similar macrocyclic structures that have been modified to enhance their therapeutic efficacy. Examples of FK506-like compounds include, for example, those described in WO 00101385. In some embodiments, the term "rapamycin compounds" as used herein does not include FK506-like compounds.

[0147] 2. Anti-inflammatory drugs Other suitable therapeutic agents include, but are not limited to, anti-inflammatory agents. The anti-inflammatory agent may be a non-steroid, a steroid, or a combination thereof. One embodiment provides an oral composition containing about 1% (w / w) to about 5% (w / w), typically about 2.5% (w / w) of an anti-inflammatory agent. Representative examples of nonsteroidal anti-inflammatory drugs include, but are not limited to, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, such as aspirin, disalcid, benorylate, trilysate, safapryn, solprin, diflunisal, and fendosal; acetates, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acemetacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac. Derivatives; fenamic acids such as mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid and tolfenamic acid; propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miloprofen, tioxaprofen, suprofen, alminoprofen and tiaprofenic acid; pyrazoles such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone and trimethasone. Mixtures of these nonsteroidal anti-inflammatory drugs may also be used.

[0148] Representative examples of steroidal anti-inflammatory drugs include, but are not limited to, hydrocortisone, hydroxyl-triamcinolone, alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorone acetonide, fludrocortisone, flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortine butyl ester, flucortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, coumadin, Corticosteroids such as lutodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflurosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and its ester balance, chloroprednisone, chloroprednisone acetate, clocortelone, clesinolone, dichlorisone, diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof. EXAMPLES

[0149] method: Synthesis of CKK-E12: Compound 1 (20 g, 41.9 mmol) was added to a 100 ml flask and trifluoroacetic acid (42 mL) was added slowly at 0° C., then stirred at room temperature for 30 min. The solvent was evaporated under reduced pressure, and the crude product dissolved in DMF (5 mL) was then added dropwise to pyridine (300 mL) at 0° C. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure to give a white solid, which was washed with ethyl acetate to give compound 2 (8.4 g, 13.04 mmol, 31% yield) (FIG. 6B). Pd / C (10 wt%, 3.0 g) was added to a solution of compound 2 in acetic acid / CHCl (150 / 150 mL). The black suspension was degassed with hydrogen for 5 min and stirred at room temperature under hydrogen atmosphere overnight. The reaction mixture was filtered through celite and washed with MeOH. The combined filtrate was concentrated to give a crude yellow viscous oil. The oil was solidified by adding ethyl acetate and washed with ethyl acetate to give compound 3 (FIG. 6C). Triethylamine (0.12 mL, 0.88 mmol) was added to a solution of compound 3 (84 mg, 0.22 mmol) and 1,2-epoxydodecane (247 mg, 1.34 mmol) in EtOH (2 mL). After stirring at room temperature for 30 min, the reaction mixture was then irradiated in a microwave reactor at 150° C. for 5 h. The crude residue was purified via flash column chromatography. The version with four alkyl tails was purified using flash column chromatography and then the chemical structure was confirmed using 1H-NMR (FIG. 6C).

[0150] Nanoparticle Formulation: Nanoparticles were formulated in a microfluidic device by mixing Cre mRNA, DNA, ionizable lipids, PEG, and cholesterol as previously described (Poisson, et al., Journal of Hepatology, 66:212 (2017)). The experimental design can be visualized in Figure 1H. Nanoparticles were prepared using variable molar ratios of these components. Nucleic acids (e.g., DNA barcode, mRNA) were diluted in 10 mM citrate buffer (Teknova) and loaded into a syringe (Hamilton Company). The materials that make up the nanoparticles (CKK-E12, cholesterol, PEG, DOPE) were diluted in ethanol and loaded into a second syringe. The citrate and ethanol phases were mixed together in the microfluidic device using a syringe pump.

[0151] DNA barcoding: Methods for screening and formulating nanoparticles using DNA barcodes are described in PCT / US2018 / 058171. Each chemically distinct LNP was formulated to have its own distinct DNA barcode. For example, LNP1 had Cre mRNA and DNA barcode1, while chemically distinct LNP2 had Cre mRNA and DNA barcode2. The DNA barcodes were rationally designed with universal primer sites and specific 8-nucleotide barcode sequences, similar to those previously described (Paunovaska, K., et al. Nano Lett, 18:2148 (2018)). Universal forward and reverse primer regions were included in all barcodes to ensure equal amplification of each sequence. The 8-nt sequences were 4 8 It can generate over (65536) different barcodes.

[0152] Nanoparticle characterization: Dynamic light scattering (DLS) was used to measure LNP hydrodynamic diameter. LNPs were diluted in sterile 1x PBS to a concentration of approximately 0.06 μg / mL and analyzed. LNPs were included if they met three criteria: diameter greater than 20 nm, diameter less than 215 nm, and an autocorrelation function with only one inflection point. Particles meeting these criteria were pooled, dialyzed against 1x phosphate buffered saline (PBS, Invitrogen), and sterile filtered through a 0.22 μm filter.

[0153] Animal testing: All animal experiments were performed in accordance with the Georgia Institute of Technology's Animal Care and Use Committee (IACUC). C57BL / 6J (no. 000664) and Ai14 LSL-Tomato (no. 007914) mice were purchased from Jackson Laboratory. For all experiments, mice were 5-8 weeks old and N=3-4 mice per group were injected intravenously via the tail vein. Nanoparticle concentrations were determined using a NanoDrop (Thermo Scientific). Body weights for all mice are included in Figure 7A-C.

[0154] In all cases, mice were sacrificed 3 days after administration of LNPs and immediately perfused with 20 mL of 1x PBS through the right atrium. Organs were isolated immediately after perfusion. Tissues were cut and then placed in a digestive enzyme solution containing collagenase type I (Sigma Aldrich), collagenase XI (Sigma Aldrich) and hyaluronidase (Sigma Aldrich) for 45 min at 37°C and 750 rpm. Digestive enzymes for heart and spleen included collagenase IV (Sigma Aldrich). Digested tissues were passed through a 70 μm filter to lyse red blood cells. Cells were stained to identify specific cell populations and sorted using a BD FacsFusion cell sorter. The antibody clones used for staining were anti-CD31 (390, BioLegend), anti-CD45.2 (104, BioLegend), anti-CD11b (M1 / 70, BioLegend), anti-CD68 (FA11, BioLegend), anti-CD3 (17A2, BioLegend), anti-CD19 (6D5, BioLegend), and anti-CD34 (SA376A4, BioLegend). Table 1 shows the FACS markers used for each cell and tissue type.

[0155] [Table 1]

[0156] PCR amplification: All samples were amplified and prepared for sequencing using a nested PCR protocol. More specifically, 1 μL of each primer (10 μM reverse / forward) was added to 5 μL Kapa HiFi 2x master mix, 2 μL sterile H2O, and 1 μL DNA template. The second PCR added the Nextera XT chemistry index and i5 / i7 adapter regions and used the product from "PCR1" as template.

[0157] Deep Sequencing: Illumina deep sequencing was performed on an Illumina MiniSeq™ using standard protocols proposed by Illumina. Sequencing was performed at the Georgia Tech Molecular Evolution Core.

[0158] Data normalization: Counts for each particle were normalized to the barcoded LNP mixture injected into the mice, as we previously described (Paunovaska, K., et al. Nano Lett, 18:2148 (2018)).

[0159] Data Analysis: Sequencing results were processed using a custom Phython-based tool to extract raw barcode counts for each tissue. These raw counts were then normalized with an R script before further analysis. Statistical analysis was performed using GraphPad Prism 7. Data are plotted as mean ± standard error unless otherwise stated.

[0160] [Example 1] Synthesis of a library of rationally designed nanoparticles containing oxidized cholesterol. result: As a quality control, the size of each individual LNP was analyzed. LNPs were pooled only if their hydrodynamic diameter was between 20 and 215 nm and their autocorrelation curve contained one inflection point (Figure 4A-Figure 4B). 86 of the 125 formulated LNPs met these criteria and were therefore pooled. As a control, the diameters of all 86 LNPs were compared to the diameter of the pooled LNP solution and found to be similar (Figure 2C). This suggests that the pooled LNPs did not aggregate after mixing. Naked DNA barcodes were added as a negative control, as naked DNA does not readily enter cells. After isolating cells and performing NGS, it was determined that, as expected, naked DNA was not delivered to cells as frequently as the total DNA barcode delivered by LNPs (Figure 2D-Figure 2E).

[0161] Mice were sacrificed 72 hours after injection of pooled LNPs. This time point allows cells to express tdTomato after Cre mRNA delivery (Figure 2A). Liver, spleen, heart, kidney, pancreas, lung and bone marrow were then isolated. Using fluorescence activated cell sorting (FACS), 28 distinct tdTomato+ cell types were isolated (Figure 2A). Comparing the percentage of tdTomato+ cell types in the various organs, cells in the liver tended to be more targeted than cells in the other organs (Figure 2B). The organ with the second highest percentage of tdTomato+ cells was the spleen. The remaining five organs had negligible delivery (Figure 4C). An unbiased Euclidean algorithm was used to cluster the barcode sequencing data. This bioinformatics technique can be systematically applied to gene expression data to analyze nanoparticle barcode data. Euclidean clustering revealed that the four liver cell types tended to "cluster" more closely together than the spleen cell types (Figure 2F). We found that the percentages of tdTomato+ hepatic endothelial cells, hepatic immune cells, and Kupffer cells were much higher than the percentage of tdTomato+ hepatocytes (Figure 2B).

[0162] [Example 2] Modified cholesterol can alter nanoparticle delivery in vivo method: Unmodified cholesterol was subjected to enzymatic action to form variants that were side-chain or ring oxidized (Figure 1A). To investigate whether these modifications altered LNP targeting, 125 LNPs were formulated using microfluidics (Figure 1B-G). To minimize variability from components other than cholesterol, LNPs were made from the ionizable lipid-like material cKK-E12, two well-validated PEG-lipids, the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and one of nine different cholesterol variants (Figure 1B-G).

[0163] LNPs were formed by combining and mixing the contents in a microfluidic device. Each LNP had a unique DNA barcode that acted as a tag for that LNP, as well as Cre mRNA to signal functional mRNA delivery (Figure 1H). Stable LNPs were pooled together (Figure 1I) and administered to Ai14 mice at a total nucleic acid dose of 1.0 mg / kg. Ai14 mice contain a Lox-Stop-Lox-tdTomato construct under the control of a CAG promoter; as a result, cells in Ai14 mice become tdTomato+ once (1) Cre mRNA is delivered to the cytoplasm, (2) Cre mRNA is translated into Cre protein, (3) Cre protein translocates from the cytoplasm to the nucleus, and (4) Cre protein edits the genome by removing the "Stop" between the Lox sites. Thus, by isolating tdTomato+ cell types using fluorescence-activated cell sorting (FACS) and quantifying the barcodes therein using next-generation sequencing (NGS), we can identify LNPs located in cells where functional mRNA delivery has occurred (Figure 1I). NGS sequencing data was quantified as "normalized delivery," similar to counts per million in RNA-seq (Figure 1I).

[0164] result: To quantify the impact of cholesterol structure on overall spleen and liver delivery, we calculated normalized delivery for all eight cell types sequenced (four in spleen and four in liver) (Figure 3A). Side-chain oxidized cholesterol variants tended to enhance delivery compared to other cholesterol variants (Figure 3B-C). Notably, LNPs formulated with 25-hydroxycholesterol (25-OH) and 20α-hydroxycholesterol (20α-OH) resulted in higher normalized delivery across all eight cell types (Figure 3D). To complement these calculations, we determined which cholesterol variants were enriched in the top 10% of LNPs (Figure 3E). We then calculated the enrichment in the bottom 10% of LNPs and subtracted it from the enrichment in the top 10% (Figure 3E-F). This identifies how likely a given cholesterol variant is to be found in the best and worst performing LNPs. The enrichment calculations are detailed in Figure 5A. As an additional control, the same two analyses were performed for cholesterol mole percent, ionized lipid mole percent, and phospholipid mole percent - normalized delivery and enrichment across all eight cell types (Figure 5B-D). No significant trends were observed.

[0165] These data suggested that LNP cholesterol chemical composition is an important factor in LNP targeting. However, this did not take into account the size of the LNP. We previously found no relationship between nanoparticle size and delivery for hydrodynamic diameters between about 20 and about 200 nm. To explore how much size altered delivery in this experiment, we calculated whether normalized delivery varied with LNP size for all LNPs (Figure 5K), LNPs with tail oxidized cholesterol (Figure 5L), and LNPs with ring oxidized cholesterol (Figure 5M); no relationship was found. We calculated the percentage of formulated LNPs that met the inclusion criteria as a function of cholesterol structure and the average size of stable LNPs based on cholesterol structure. No significant differences were found (Figure 5N-O). The same analysis was performed as a function of cholesterol mole percent, reaching the same conclusion (Figure 5P-Q). Thus, there was no evidence that size affected LNP delivery between 20 and 215 nm.

[0166] To this end, the top three LNP candidates identified were formulated with Cre mRNA. Of the three formulated LNPs, one LNP that contained 25-OH cholesterol did not formulate consistently and was therefore excluded. The remaining two LNPs that contained 20α-OH cholesterol (Figure 3G-K) formed stable LNPs and were therefore administered to Ai14 mice at a total mRNA dose of 0.25 mg / kg. Encouragingly, both LNPs reproduced the results from the previous screen (Figure 3L-O). Both LNP1 and LNP2 robustly targeted cells in the microenvironment following injection at 0.25 mg / kg. As predicted by the screen, hepatocytes were targeted much less efficiently. Encouraged by the robust delivery at 0.25 mg / kg, LNP1 and LNP2 were injected at a dose of 0.05 mg / kg. Once again, robust delivery was observed (Figure 3L-O). Both LNP1 and LNP2 worked equally well at 0.25 mg / kg by saturating the Cre-based system, but at the lower dose of 0.05 mg / kg, LNP1 outperformed LNP2. LNP1 did not cause mice to lose weight in any experiment; LNP2 caused mice to lose weight at 0.25 mg / kg (Figure 7A-C).

[0167] Systemic delivery of therapeutic RNA to hepatocytes has resulted in FDA-approved drugs (Rizk, M and Tuzmen, S., Pharmacogenomics and Personalized Medicine, 10:267 (2017)). Delivery to non-hepatocytes has remained more challenging. Thus, an unbiased, high-throughput method to study how LNPs deliver RNA in vivo could accelerate the discovery of nanoparticles with novel tropism. Here, we report that LNPs containing oxidized cholesterol can deliver mRNA to cells in the liver microenvironment more potently than to hepatocytes. Notably, robust delivery occurred at 0.05 mg / kg, below the dosing regime used for siRNA therapy in humans. Given the importance of liver endothelial cells (Poisson, et al., Journal of Hepatology, 66:212 (2017)) and Kupffer cells (Kolios, G., et al., World Journal of Gastroenterology, 12:7413 (2006)) in disease, these data suggest that further advances in delivery may ultimately result in protein replacement therapy within the liver microenvironment.

[0168] In the foregoing specification, the invention has been described with respect to certain embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments, and that some of the details described herein may be modified considerably without departing from the underlying principles of the invention.

[0169] All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from its spirit or essential attributes, and therefore, reference should be made to the appended claims, rather than the foregoing specification, as indicating the scope of the invention. The present invention also includes the following aspects. <1> An ionized lipid; Phospholipids and Polyethylene glycol, Oxidized cholesterol and A lipid nanoparticle composition comprising: <2> 2. The nanoparticle composition of claim 1, wherein the ionizable lipid is 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (CKK-E12). <3> 3. The nanoparticle composition according to claim 1 or 2, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). <4> The PEG is C 14 PEG 2000 or C 18 PEG 2000 4. The nanoparticle composition according to any one of 1 to 3 above, <5> 5. The nanoparticle composition according to any one of claims 1 to 4, wherein the oxidized cholesterol is modified with a hydroxyl group near the D sterol ring. <6> 5. The nanoparticle composition described in any one of 1 to 4 above, wherein the oxidized cholesterol is selected from the group consisting of 20α-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 7β,25-dihydroxylcholesterol, 27-hydroxycholesterol, 25-hydroxycholesterol, or a combination thereof. <7> 7. The nanoparticle composition according to any one of 1 to 6 above, comprising about 30 mol% to about 80 mol% ionized lipid, about 5 mol% to about 55 mol% cholesterol, about 10 mol% to about 35 mol% phospholipid, and about 0 mol% to about 20 mol% PEG-lipid. <8> 8. The nanoparticle composition according to any one of 1 to 7 above, further comprising a therapeutic or prophylactic agent. <9> 9. The nanoparticle composition described in claim 8, wherein the therapeutic or prophylactic agent is encapsulated within a lipid nanoparticle. <10> 9. The nanoparticle composition according to claim 8, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA). <11> 11. The nanoparticle composition of claim 10, wherein the RNA is messenger RNA (mRNA). <12> 12. The nanoparticle composition according to any one of 1 to 11 above, having a diameter of about 20 nm to about 215 nm. <13> A pharmaceutical composition comprising the lipid nanoparticles described in any one of 1 to 12 above and a pharma- ceutically acceptable excipient. <14> A method for delivering a therapeutic or prophylactic agent to a subject in need thereof, comprising administering to the subject a lipid nanoparticle composition described in any one of 8 to 12 above. <15> 15. The method of claim 14, further comprising administering a second therapeutic agent. <16> 15. The method of claim 14, wherein the nanoparticles deliver cargo preferentially to cells in the liver. <17> 17. The method of claim 16, wherein the liver cells include Kupffer cells and endothelial cells. <18> A method for preventing or treating liver disease in a subject in need thereof, comprising administering to the subject an effective amount of lipid nanoparticles described in any one of 1 to 12 above to prevent or treat liver disease. <19> 16. The method according to claim 15, wherein the amount of lipid nanoparticles administered to the subject is 0.05 mg / kg. <20> 16. The method according to claim 15, wherein the liver disease is selected from the group consisting of liver cancer, liver cirrhosis, non-alcoholic fatty liver disease, and alcohol-related liver disease. <21> 1. A method for delivering a drug to Kupffer cells in a subject in need thereof, comprising: Administering the lipid nanoparticles according to any one of claims 8 to 12 in an amount effective to deliver the drug to the Kupffer cells. The method includes: <22> 1. A method for delivering a drug to liver endothelial cells in a subject in need thereof, comprising: Administering the lipid nanoparticles according to any one of claims 8 to 12 in an amount effective to deliver the drug to the liver endothelial cells. The method includes: <23> 1. A method for delivering a drug to an immune cell of a subject in need thereof, comprising: Administering the lipid nanoparticles according to any one of claims 8 to 12 in an amount effective to deliver the drug to the liver endothelial cells. The method includes: <24> CKK-E12 and DOPE and C 18 PEG 2000 and, 20α-hydroxycholesterol and A lipid nanoparticle composition comprising: <25> CKK-E12 and DOPE and C 18 PEG 2000 and, 25-hydroxycholesterol and A lipid nanoparticle composition comprising: <26> A lipid nanoparticle composition described in any of 1 to 12 or 24 to 25 above, or a pharmaceutical composition described in 13 above, wherein the ionizable lipid, the phospholipid, the polyethylene glycol and the oxidized cholesterol are all selected in combination together to obtain a lipid nanoparticle composition having the ability to more potently deliver mRNA encapsulated within the nanoparticles to non-hepatic cells than to hepatic cells in the liver microenvironment.

Claims

1. An ionized lipid; Phospholipids and Polyethylene glycol-lipid; Oxidized cholesterol and A lipid nanoparticle composition comprising: The lipid nanoparticle composition, wherein the oxidized cholesterol is selected from the group consisting of 20α-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7β-hydroxycholesterol, and combinations thereof.

2. 2. The nanoparticle composition of claim 1, wherein the ionizable lipid is 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (CKK-E12).

3. The nanoparticle composition of claim 1 or 2, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

4. The polyethylene glycol-lipid is C １４ PEG ２０００ Or C １８ PEG ２０００ The nanoparticle composition of claim 1 , wherein

5. The nanoparticle composition of claim 1 , wherein the oxidized cholesterol is 20α-hydroxycholesterol.

6. The nanoparticle composition of claim 1 , wherein the oxidized cholesterol is 25-hydroxycholesterol.

7. The nanoparticle composition of claim 1 , wherein the oxidized cholesterol is 7β-hydroxycholesterol.

8. The nanoparticle composition of claim 1 , wherein the oxidized cholesterol is 27-hydroxycholesterol.

9. 9. The nanoparticle composition of any one of claims 1 to 8, comprising 30 mol% to 80 mol% of ionized lipid, 5 mol% to 55 mol% of oxidized cholesterol, 10 mol% to 35 mol% of phospholipid, and 5 mol% to 20 mol% of polyethylene glycol-lipid.

10. The nanoparticle composition of claim 1 , further comprising a therapeutic or prophylactic agent.

11. The nanoparticle composition of claim 10, wherein the therapeutic or prophylactic agent is encapsulated as cargo within the lipid nanoparticle.

12. The nanoparticle composition of claim 10 , wherein the therapeutic or prophylactic agent is a ribonucleic acid (RNA).

13. The nanoparticle composition of claim 12 , wherein the RNA is messenger RNA (mRNA).

14. The nanoparticle composition of any one of claims 1 to 13, having a diameter of from 20 nm to 215 nm.

15. A pharmaceutical composition comprising the lipid nanoparticle composition of any one of claims 1 to 14 and a pharma- ceutically acceptable excipient.

16. 15. A pharmaceutical composition for delivering a therapeutic or prophylactic agent comprising a lipid nanoparticle composition according to any one of claims 10 to 14, 15. The pharmaceutical composition for use in a method for delivering a therapeutic or prophylactic agent to a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle composition described in any one of claims 10 to 14.

17. 17. The pharmaceutical composition of claim 16, wherein the method further comprises administering a second therapeutic agent.

18. 17. The pharmaceutical composition of claim 16, wherein the nanoparticles deliver cargo preferentially to cells in the liver.

19. 19. The pharmaceutical composition of claim 18, wherein the cells in the liver include Kupffer cells and endothelial cells.

20. A pharmaceutical composition for preventing or treating liver disease comprising a lipid nanoparticle composition according to any one of claims 1 to 14, The pharmaceutical composition for use in a method for preventing or treating liver disease in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle composition described in any one of claims 1 to 14 in an amount effective to prevent or treat liver disease.

21. 21. The pharmaceutical composition of claim 20, wherein the amount of lipid nanoparticles administered to the subject is 0.05 mg / kg.

22. 21. The pharmaceutical composition of claim 20, wherein the liver disease is selected from the group consisting of liver cancer, liver cirrhosis, non-alcoholic fatty liver disease, and alcohol-related liver disease.

23. A pharmaceutical composition for delivering a therapeutic or prophylactic agent to Kupffer cells, comprising the lipid nanoparticle composition of any one of claims 10 to 14, The pharmaceutical composition for use in a method for delivering a therapeutic or prophylactic agent to Kupffer cells of a subject in need thereof, the method comprising administering an effective amount of a lipid nanoparticle composition described in any one of claims 10 to 14 to deliver the therapeutic or prophylactic agent to the Kupffer cells.

24. A pharmaceutical composition for delivering a therapeutic or prophylactic agent to liver endothelial cells, comprising the lipid nanoparticle composition of any one of claims 10 to 14, The pharmaceutical composition for use in a method for delivering a therapeutic or prophylactic agent to liver endothelial cells of a subject in need thereof, the method comprising administering an effective amount of a lipid nanoparticle composition described in any one of claims 10 to 14 to deliver the therapeutic or prophylactic agent to the liver endothelial cells.

25. A pharmaceutical composition for delivering a therapeutic or prophylactic agent to an immune cell comprising a lipid nanoparticle composition according to any one of claims 10 to 14, The pharmaceutical composition for use in a method for delivering a therapeutic or prophylactic agent to immune cells of a subject in need thereof, the method comprising administering a lipid nanoparticle composition described in any one of claims 10 to 14 in an amount effective to deliver the therapeutic or prophylactic agent to liver endothelial cells.

26. 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (CKK-E12), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), C １８ PEG ２０００ and, 20α-hydroxycholesterol and A lipid nanoparticle composition comprising:

27. 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (CKK-E12), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), C １８ PEG ２０００ and, 25-hydroxycholesterol and A lipid nanoparticle composition comprising:

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