Nucleic acid delivery vector targeting liver parenchymal cells and use thereof

Abstract

The present invention relates to a material for local drug delivery and use thereof. Specifically provided is a nanoparticle composition. The nanoparticle composition comprises a cationic lipid, a PEG lipid, and a structural lipid, wherein the lipid component of the PEG lipid is 1.0-5.5 mol%, or a range between any two of the described values. The nanoparticle composition of the present invention is delivered only at the site of administration.

Description

Nucleic acid delivery vector targeting liver parenchymal cells and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a nucleic acid delivery vector targeting liver parenchymal cells and application thereof BACKGROUND

[0002] Hepatolenticular degeneration (HLD), also known as Wilson's disease (WD), is a severe autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene located on chromosome 13. The ATP7B gene encodes a copper-dependent P-type ATPase that transports copper to the Golgi apparatus, where it is incorporated into ceruloplasmin (CP) and excreted in bile. Mutations in this gene disrupt the normal structure and function of the ATPase, leading to reduced biliary excretion of copper ions, which accumulate in the liver and can be secreted into the blood, depositing in organs and tissues such as the brain, kidneys, and cornea, resulting in liver damage, central nervous system abnormalities, and psychiatric symptoms. Previous foreign reports have shown that the incidence of WD is 1 / 100,000 to 1 / 30,000. China currently lacks large-sample multi-center investigations of WD incidence, but some literature analysis suggests that the disease incidence in China is higher than in Western countries. WD can occur at any age, but is more common in children and adolescents. The most common clinical manifestations are cirrhosis (45%), neurological symptoms (35%), and psychiatric symptoms (10%).

[0003] WD is one of the few genetic diseases with effective treatment methods, but there is no effective cure. Therefore, once diagnosed, WD requires lifelong treatment. In addition to attention to copper intake in the diet, drug therapy is currently the main treatment for WD patients. There are two main categories of treatment drugs: one is metal chelators, which bind to copper in the blood and tissues, thereby removing excess copper from the body, such as penicillamine, trientine, dimercaprol sodium, and dimercaptosuccinic acid; the second is to compete with copper absorption in the intestine to reduce dietary copper intake, such as zinc agents and traditional Chinese medicine liver bean tablets. Compared with most single-gene genetic diseases, WD patients can achieve better prognosis through early diagnosis and treatment. However, this disease is a genetic disease, with a small age of onset and a need for lifelong treatment once diagnosed. About 30% to 50% of WD patients have experienced non-compliance during medication, and some patients have developed irreversible neuropsychiatric symptoms. For patients with decompensated cirrhosis who do not respond well to drug therapy, liver transplantation can achieve better prognosis, but liver transplant patients require lifelong immunosuppressive therapy. Hepatocyte transplantation and gene therapy are also limited by immune rejection and are not widely used in clinical practice. SUMMARY

[0004] In view of this huge unmet clinical need, the present application constructs a new type of ATP7B mRNA drug and its delivery carrier, which is targeted by liver parenchymal cells after intravenous injection and replaces the original ATP7B protein of the organism to restore the original copper metabolism function of the organism, so that most WD patients, especially those copper chelators / absorption inhibitors drug-resistant and poor liver transplantation treatment effect patients achieve better prognosis.

[0005] The first application of the present application provides a nanoparticle composition, which comprises a cationic lipid, a PEG lipid and a structural lipid; wherein the lipid component of the PEG lipid is 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 mol%, or a range between any two of the above values.

[0006] In one or more embodiments, the nanoparticle composition further comprises a therapeutic agent and / or a prophylactic agent.

[0007] In one or more embodiments, the cationic lipid is selected from one or more of DLin-KC2-DMA, D-Lin-MC3-DMA, DOTMA, DOTAP, DODAP, DOP-DEDA, DODMA, DC-Chol, D-Lin-MC3-DMA and SM-102.

[0008] In one or more embodiments, the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof; preferably, the PEG lipid is selected from one or more of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid; preferably, the lipid is selected from one or more of PEG-DMG, OH-PEG-DMG, PEG-DSPE, PEG-DSG, PEG-dipalmityl, PEG-dioleyl, PEG-distearyl, PEG-DAG, PEG-DPPE, PEG-c-DMA or DMG-PEG 2000 ; more preferably, the PEG lipid is OH-PEG 2000 -DMG.

[0009] In one or more embodiments, the phospholipid is the lipid of (I);

[0010] wherein Rp represents a phospholipid moiety, and R1and R2represent fatty acid moieties with or without unsaturation, which can be the same or different. The phospholipid moiety can be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moieties can be selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, a-linolenic acid, erucic acid, phytanic acid, eicosanoic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0011] In one or more embodiments, the phospholipid is selected from the group consisting of DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, DUPC, POPC, OChemsPC, DOPG, and sphingomyelin.

[0012] In one or more embodiments, the structural lipid is selected from one or more of Cholesterol, Dehydrocholesterol, Dihydrocholesterol, Desmosterol, Coprostanol, Sitosterol, β-Sitosterol, Ergocalciferol, Dihydroergocalciferol, Cholestanol, Ergosterol, Dihydroergosterol, Melitoasterol, Epicholesterol, Fucosterol, Hexahydrofarnesol, Hydroxycholesterol, Campesterol, Stigmasterol, Stigmastanol, Tomato alkaloid, Tomato alkaloid, Cholic acid, Ursolic acid, a-Tocopherol, Glycocholic acid, Taurocholic acid, Deoxycholic acid, and Lithocholic acid; preferably, the structural lipid is Cholesterol; preferably, the Cholesterol is a polypeptide-modified Cholesterol; preferably, the structural lipid comprises Cholesterol and a corticosteroid or a combination thereof.

[0013] The second aspect of the application provides a pharmaceutical composition comprising the nanoparticle composition and a pharmaceutically acceptable excipient, which is a locally acting pharmaceutical composition.

[0014] In one or more embodiments, the therapeutic agent and / or prophylactic agent is an RNA.

[0015] In one or more embodiments, the RNA is selected from the group consisting of a short chain, an antagonist, an antisense strand, a ribozyme, a small interfering RNA (siRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA, a small hairpin RNA (shRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), and a mixture thereof.

[0016] In one or more embodiments, the mRNA is transcribed from an ATP7B gene.

[0017] In one or more embodiments, the coding sequence of the mRNA is set forth in SEQ ID NO: 1.

[0018] The third aspect of the application provides a method of delivering a therapeutic and / or prophylactic agent to a liver parenchymal cell, the method comprising the step of administering (e.g., injecting) to a subject a nanoparticle composition comprising: (1) a lipid component comprising a cationic lipid, a phospholipid, a PEG lipid, and a structural lipid, and (2) a therapeutic and / or prophylactic agent, wherein the administering comprises contacting the liver parenchymal cell with the nanoparticle composition, thereby delivering the therapeutic and / or prophylactic agent to the liver parenchymal cell; wherein the lipid component of the PEG lipid is 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 mol%, or a range between any two of the foregoing.

[0019] Preferably, the lipid component of the PEG lipid is 1.0 mol% to 5.0 mol%.

[0020] Preferably, the lipid component of the PEG lipid is 1.5 mol% to 5.0 mol%.

[0021] Preferably, the lipid component of the PEG lipid is 1.5 mol% to 3.0 mol%.

[0022] Preferably, the lipid component of the PEG lipid is 3.0 mol% to 5.0 mol%.

[0023] Preferably, the lipid component of the PEG lipid is 2.0 mol% to 4.5 mol%.

[0024] In one or more embodiments, the subject is a mammal, e.g., a human.

[0025] The fourth aspect of the application provides a method of producing a polypeptide in a liver parenchymal cell, the method comprising the step of contacting the liver parenchymal cell with a nanoparticle composition comprising: (1) a lipid component comprising a cationic lipid, a phospholipid, a PEG lipid, and a structural lipid, and (2) an mRNA encoding the polypeptide, from which the polypeptide can be translated in the liver parenchymal cell to produce the polypeptide.

[0026] The fifth aspect of the application provides a method of treating a disease or condition in a mammal (e.g., a human) by administering a therapeutic and / or prophylactic agent to a liver parenchymal (but not systemic) cell, or to locally effect a therapeutic and / or prophylactic agent, the method comprising the step of administering (e.g., injecting) to a liver parenchymal cell of the mammal a therapeutically effective amount of a nanoparticle composition comprising: (1) a lipid component comprising a cationic lipid, a phospholipid, a PEG lipid, and a structural lipid, and (2) a therapeutic and / or prophylactic agent (e.g., an mRNA).

[0027] In one or more embodiments, the disease or condition is a disease or condition of dysregulation or abnormality of a protein or polypeptide.

[0028] In one or more embodiments, the disease is a disease that benefits from delivery of a therapeutic and / or prophylactic agent to hepatocytes.

[0029] In one or more embodiments, the disease or condition is selected from rare diseases, infectious diseases, cancers and proliferative diseases, genetic diseases (e.g. cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases.

[0030] In one or more embodiments, the disease or condition is Wilson’s disease.

[0031] The sixth aspect of the application provides use of the nanoparticle composition and / or pharmaceutical composition as described in any embodiment herein in the manufacture of a medicament for the treatment or prevention of a disease.

[0032] In one or more embodiments, the disease is a disease that benefits from delivery of a therapeutic and / or prophylactic agent to hepatocytes.

[0033] In one or more embodiments, the disease is Wilson’s disease.

[0034] The seventh aspect of the application provides a method of treatment of Wilson’s disease comprising providing to a subject suffering from Wilson’s disease a pharmaceutical composition as described in any embodiment herein.

[0035] The application also provides a method of making the nanoparticle composition described herein. The method comprises mixing (e.g. microfluidising) the components of the nanoparticle composition. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is a graph of particle size distribution, encapsulation efficiency and polydispersity index for different LNP carriers. Figure 1(A) is a graph of particle size distribution for 1.5% -OH, 3% -OH, 5% -OH. Figure 1(B) is a table of specific information for LNP.

[0037] Figure 2 is the EGFP protein expression of liver parenchymal cells and nonparenchymal cells at 8h, 24h after intravenous injection of 1.5%-OH ATP7B mRNA / LNP, 3%-OH ATP7B mRNA / LNP and 5%-OH ATP7B mRNA / LNP. Figure 2(A) is the EGFP protein expression of liver parenchymal cells in each experimental group; Figure 2(B) is the EGFP protein expression of liver T cells. Figure 2(C) is the EGFP protein expression of liver B cells; Figure 2(D) is the EGFP protein expression of liver macrophages. Compared with 1.5%-OH LNP and 5%-OH LNP, 3%-OH LNP can improve the EGFP protein expression of liver parenchymal cells, and the effect is most obvious at 8h, and the average fluorescence intensity is about twice that of the other two experimental groups, while reducing the EGFP protein expression of liver B cells and T cells.

[0038] Figure 3 is the expression effect of ATP7B protein at 24h after transfection of Huh7 cells with 3%-OH ATP7B mRNA / LNP.

[0039] Figure 4 is the structure of copper concentration-responsive LUC plasmid and the copper excretion function of cells.

[0040] Figure 5 is the expression effect of liver ATP7B protein of Wilson's disease mice after tail vein injection of 3%-OH ATP7B mRNA / LNP.

[0041] Figure 6 is the results of liver H&E section, liver copper content and urine copper content of Wilson's disease mice after tail vein injection of 3%-OH ATP7B mRNA / LNP. DETAILED DESCRIPTION

[0042] In order for those skilled in the art to understand the characteristics and effects of the present application, the following is a general description and definition of the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used in the text are the usual meanings understood by those skilled in the art for the present application, and in case of conflict, the definition in the specification shall prevail.

[0043] Theories or mechanisms described and disclosed herein, whether correct or not, should not be considered limiting the scope of the present application, i.e., the present application can be practiced without any particular theory or mechanism.

[0044] The techniques and procedures described or referenced herein include techniques and procedures that are generally well understood and / or routinely practiced by those skilled in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003).

[0045] In this document, "include," "includes," "including," "contain," "containing," "contains," and the like are inclusive and meant to be equivalent to "comprising," "comprises," "comprising," and the like.

[0046] In this document, all features that are described in terms of a numerical range or a percentage range are intended to be merely illustrative and convenient. Thus, the description of a numerical range or a percentage range is intended to be construed as having encompassed and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0047] In this document, unless otherwise specified, percentages are mass percentages and ratios are mass ratios.

[0048] In this document, the sum of the percentages of the components of a composition is 100%.

[0049] In this document, when describing embodiments or examples, it is understood that the embodiments or examples are not intended to limit the application to those embodiments or examples. On the contrary, all alternatives, modifications, and equivalents of the methods and materials described herein that are within the scope of the claims are to be included.

[0050] In this document, for the sake of brevity, not all possible combinations of the various features described in the various embodiments or examples are described. Thus, any combination of the various features described in the various embodiments or examples is possible, as long as the combination is not contradictory, and all possible combinations are to be considered as being within the scope of the present specification.

[0051] The inventors have found, through extensive and in-depth research, that by changing the ratio between the components of the nucleic acid delivery carrier, increasing the proportion of the PEG lipid component, a nucleic acid delivery carrier for targeting the expression of a target protein in liver parenchymal cells can be constructed. The nucleic acid delivery carrier is preferentially delivered to liver parenchymal cells, and the nucleic acid carried by the nucleic acid delivery carrier has an expression advantage in liver parenchymal cells. The nucleic acid delivery carrier significantly improves the expression of the target protein in liver parenchymal cells and reduces the expression of the target protein in liver non-parenchymal cells (such as immune cells). Meanwhile, the delivery carrier loaded with ATP7B mRNA (SEQ ID NO: 1) is successfully verified to have the effect of reducing the copper content in the liver of a mouse model of hepatolenticular degeneration. Thus, the present application is completed.

[0052] The present disclosure relates to a new method of formulating a lipid nanoparticle composition, and also provides methods of delivering a therapeutic and / or prophylactic agent to a mammalian cell, in particular, to a local region of a mammal, producing a polypeptide of interest in a cell of the local region of the mammal, and treating a disease or condition of the mammal.

[0053] The method of delivering a therapeutic and / or prophylactic agent to a mammalian cell or local region involves administering to a subject a nanoparticle composition comprising the therapeutic and / or prophylactic agent, wherein the administration involves contacting the cell or local region with the composition, whereby the therapeutic and / or prophylactic agent is delivered to the cell or local region.

[0054] As used herein, "local" refers to cells, tissues, or organs within a certain range of the site of administration. In the art, it is often necessary to use special dosage forms to retain the drug at the site of application, such as creams, ointments, sprays, and powders, and are often used on the body surface or mucosal surfaces. The inventors have discovered that using the lipid nanoparticle compositions described herein can achieve local administration within an organ, tissue, or cell with local effects. In some embodiments, the nanoparticle compositions of the present application are suitable for use in the preparation of locally applied, locally acting products (LALAPs). As used herein, "locally acting product," "locally acting product composition," "locally applied, locally acting product," or "locally applied, locally acting" refers to a product that is applied to a site and exerts its effect at the site of application, such that any systemic effects (e.g., liver targeting) are considered unintended effects of the product. Local can include only cells, only organs (e.g., liver, spleen, kidney, or lung), or only tissues (e.g., muscle, skin, or bone). In some embodiments herein, local refers to only the cells, tissues, or organs of the site of administration. In some embodiments, when a product is administered locally to an organ (e.g., liver, spleen, kidney, or lung), the product is delivered only to the organ at the site of administration; when a product is administered locally to a tissue (e.g., a muscle), the product is delivered only to the tissue (e.g., the muscle) at the site of administration; when a product is administered locally to a cell, the product is delivered only to the cell within the organ or tissue (e.g., the muscle) at the site of administration.

[0055] Lipid nanoparticle compositions

[0056] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and / or crosslinked to one another. The lipid bilayers can include one or more ligands, proteins, or channels.

[0057] The lipid component of the nanoparticle composition can include one or more positively charged or partially positively charged lipids. Such substances can alternatively be referred to as cationic lipids. Cationic lipids can form lipoplexes with negatively charged genes (DNA or RNA) through electrostatic interactions. The remaining positive charge on the surface of the cationic liposome makes the complex positively charged; the positively charged lipoplex in turn adsorbs onto the negatively charged cell surface through electrostatic interactions. The lipoplex enters the cell through endocytosis or fusion with the cell membrane; the lipoplex releases the gene in the cytoplasm or further into the nucleus for expression in the cell. The cationic lipid can be selected from one or more of DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane), D-Lin-MC3-DMA (l,2-dilinoleyl-N,N-dimethyl-3-aminopropane), dioleoylpropyltrimethylammonium chloride (DOTMA), (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-dimethylammonium- propane (DODAP), DOP-DEDA, 1,2-dioleoyl-3-dimethylamino-propane (DODMA), 3 -[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), 4-(N,N-dimethylamino)butanoic acid (dilinoleyl) methyl ester (D-Lin-MC3-DMA), and 1-octyl nonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid ester (SM-102). In some embodiments, the cationic lipid comprises SM-102. In preferred embodiments, the cationic lipid is SM-102.

[0058] The lipid component of the nanoparticle composition can include one or more PEG or PEG-modified lipids. Such substances can alternatively be referred to as pegylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipids can be selected from one or more of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the PEG lipids can be one or more of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. For example, 1,2-dimyristoyl-sn-glycero-methoxypolyethyleneglycol (PEG-DMG), hydroxyl-polyethyleneglycol (2000)-dimyristoyl-rac-glycerol (OH-PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine N-[ammonium(polyethyleneglycol)] (PEG-DSPE), PEG-disteraryl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), or DMG-PEG 2000 OH-PEG 2000 -DMG.

[0059] The lipid component of the nanoparticle composition can include one or more structural lipids. The structural lipids can be selected from, but not limited to, one or more of Cholesterol, Dehydrocholesterol, Dihydrocholesterol, Desmosterol, Coprostanol, Sitosterol, β-Sitosterol, Ergocalciferol, Dihydroergocalciferol, Cholestanol, Ergosterol, Dihydroergosterol, Melitoasterol, Epicholesterol, Fucosterol, Hexahydrofurosterol, Hydroxycholesterol, Campesterol, Stigmastanol, Stigmasterol, Brassicasterol, Spinasterol, Tomato base, Tomato base, Cholic acid, Ursolic acid, α-Tocopherol, Glycocholic acid, Taurocholic acid, Deoxycholic acid, and Lithocholic acid. In some embodiments, the structural lipid is Cholesterol. In some embodiments, the Cholesterol is a polypeptide modified Cholesterol. In some embodiments, the structural lipid includes Cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof.

[0060] The lipid component of the nanoparticle composition can include one or more phospholipids, also known as neutral lipids (helper lipids), such as one or more unsaturated lipids. The phospholipids can assemble into one or more lipid bilayers. Generally, the phospholipids can include a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid can be a lipid according to Formula (I):

[0061] wherein Rp represents a phospholipid moiety, R1and R2represent fatty acid moieties with or without unsaturation, which can be the same or different. The phospholipid moiety can be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moieties can be selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, a-linolenic acid, erucic acid, phytanic acid, eicosanoic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0062] Phospholipids useful in the compositions and methods described herein can be selected from the group consisting of DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, DUPC, POPC, OChemsPC, DOPG, and sphingomyelin. In some embodiments, the nanoparticle composition comprises DSPC. In some embodiments, the nanoparticle compositions described herein comprise SM-102, a PEG lipid, DSPC, and cholesterol. In some embodiments, the nanoparticle compositions described herein comprise SM-102, OH-PEG 2000 - DMG, DSPC, and cholesterol.

[0063] The lipid component of the nanoparticle composition can include, for example, a cationic lipid, a helper lipid, a PEG lipid, and a structural lipid as described herein. Each lipid component can be provided in a particular fraction. In certain embodiments, the lipid component of the nanoparticle composition includes about 30 mol% to about 60 mol% cationic lipid, about 1 mol% to about 5 mol% PEG lipid, about 18.5 mol% to about 48.5 mol% structural lipid, and about 0 mol% to about 30 mol% phospholipid. In some embodiments, the lipid component of the nanoparticle composition includes about 35 mol% to about 55 mol% cationic lipid, about 1.5 mol% to about 5 mol% PEG lipid, about 30 mol% to about 40 mol% structural lipid, and about 5 mol% to about 25 mol% phospholipid. In some embodiments, the lipid component of the nanoparticle composition includes about 40 mol% to about 55 mol% cationic lipid, about 1.5 mol% to about 3 mol% PEG lipid, about 35 mol% to about 40 mol% structural lipid, and about 10 mol% to about 20 mol% phospholipid. In some embodiments, the lipid component of the nanoparticle composition includes about 45 mol% to about 50 mol% cationic lipid, about 3 mol% to about 5 mol% PEG lipid, about 35 mol% to about 40 mol% structural lipid, and about 10 mol% to about 20 mol% phospholipid. In some embodiments, the phospholipid can be DOPE or DSPC. In some embodiments, the PEG lipid can be OH-PEG2000 - DMG and / or structural lipid can be cholesterol. In some embodiments, the cationic lipid is SM-102, the phospholipid is DOPE or DSPC, the PEG lipid is OH-PEG 2000 - DMG, the structural lipid is cholesterol.

[0064] In some embodiments, the PEG lipid (e.g., OH-PEG 2000 -DMG) is 1.0, 1.5, 2.0, 2.5, 2.96, 3.0, 3.5, 4.0, 4.5, 4.83, 5.0, 5.5 mol%, or a range between any two of the foregoing. In some embodiments, the lipid component of the nanoparticle composition comprises about 1 mol% to about 5.5 mol% of the PEG lipid (e.g., OH-PEG 2000 -DMG). In some embodiments, the lipid component of the nanoparticle composition comprises about 1.5 mol% to about 5.0 mol% of the PEG lipid (e.g., OH-PEG 2000 -DMG). In some embodiments, the lipid component of the nanoparticle composition comprises about 2 mol% to about 5 mol% of the PEG lipid (e.g., OH-PEG 2000 -DMG). In some embodiments, the lipid component of the nanoparticle composition comprises about 2.5 mol% to about 5 mol% of the PEG lipid (e.g., OH-PEG 2000 -DMG). In some embodiments, the lipid component of the nanoparticle composition comprises about 3 mol% to about 4.5 mol% of the PEG lipid (e.g., OH-PEG 2000 -DMG).

[0065] In some embodiments, the cationic lipid described herein (e.g., SM-102) comprises 30, 35, 40, 45, 48.3, 49.3, 50, 55, 60 mol%, or a range between any two of the foregoing, in the nanoparticle composition of the present application. In some embodiments, the structural lipid described herein (e.g., cholesterol) comprises 18.5, 20, 25, 30, 35, 37.1, 37.9, 38.5, 40, 45, 48.5 mol%, or a range between any two of the foregoing, in the nanoparticle composition of the present application. In some embodiments, the helper lipid described herein (e.g., DSPC) comprises 0, 5, 9.7, 9.9, 10, 15, 20, 25, 30 mol%, or a range between any two of the foregoing, in the nanoparticle composition of the present application.

[0066] or the lipid component can comprise, in molar fractions, for example, about 30 to about 60 molar fractions of a cationic lipid, about 1 to about 5 molar fractions of a PEG lipid (e.g., OH-PEG 2000 -DMG), about 18.5 to about 48.5 molar fractions of a structural lipid, and about 0 to about 30 molar fractions of a phospholipid. In some embodiments, the lipid component of the nanoparticle composition comprises about 35 to about 55 molar fractions of a cationic lipid, about 1.5 to about 5 molar fractions of a PEG lipid (e.g., OH-PEG 2000 -DMG), about 30 to about 40 molar fractions of a structural lipid, and about 5 to about 25 molar fractions of a phospholipid. In some embodiments, the lipid component of the nanoparticle composition comprises about 40 to about 55 molar fractions of a cationic lipid, about 1.5 to about 3 molar fractions of a PEG lipid (e.g., OH-PEG 2000 -DMG), about 35 to about 40 molar fractions of a structural lipid, and about 10 to about 20 molar fractions of a phospholipid. In some embodiments, the lipid component of the nanoparticle composition comprises about 45 to about 50 molar fractions of a cationic lipid, about 3 to about 5 molar fractions of a PEG lipid (e.g., OH-PEG 2000 -DMG), about 35 to about 40 molar fractions of a structural lipid, and about 10 to about 20 molar fractions of a phospholipid. In some embodiments, the cationic lipid is SM-102, the phospholipid is DOPE or DSPC, the PEG lipid is OH-PEG 2000 -DMG, the structural lipid is cholesterol.

[0067] In some embodiments, the lipid component of the nanoparticle composition of the present application comprises about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 molar fractions, or a range between any two of the foregoing, of a PEG lipid (e.g., OH-PEG 2000 -DMG). In some embodiments, the lipid component of the nanoparticle composition comprises about 1.5 to about 5.0 molar fractions of a PEG lipid (e.g., OH-PEG 2000 -DMG). In some embodiments, the lipid component of the nanoparticle composition comprises about 2.0 to about 5.0 molar fractions of a PEG lipid (e.g., OH-PEG 2000 -DMG). In some embodiments, the lipid component of the nanoparticle composition comprises about 2.5 to about 5.0 molar fractions of a PEG lipid (e.g., OH-PEG 2000 -DMG). In some embodiments, the lipid component of the nanoparticle composition comprises about 2.5 to about 5.0 molar fractions of a PEG lipid (e.g., OH-PEG2000 -DMG). In some embodiments, the lipid component of the nanoparticle composition comprises about 3 to about 4.5 mol% of a PEG lipid (e.g., OH-PEG 2000 -DMG).

[0068] In some embodiments, the molar percentage of a cationic lipid described herein (e.g., SM-102) in the nanoparticle compositions of the present application is 30, 35, 40, 45, 50, 55, 60 mol%, or a range between any two of the foregoing. In some embodiments, the molar percentage of a structural lipid described herein (e.g., cholesterol) in the nanoparticle compositions of the present application is 18.5, 20, 25, 30, 35, 40, 45, 48.5 mol%, or a range between any two of the foregoing. In some embodiments, the molar percentage of a helper lipid described herein (e.g., DSPC) in the nanoparticle compositions of the present application is 0, 5, 10, 15, 20, 25, 30 mol%, or a range between any two of the foregoing.

[0069] In one or more embodiments, the lipid component of the nanoparticle composition comprises about 30 to about 60 mol% of SM-102, about 1 to about 5 mol% of OH-PEG 2000 -DMG, about 18.5 to about 48.5 mol% of cholesterol, and about 0 to about 30 mol% of DSPC. In some embodiments, the lipid component of the nanoparticle composition comprises about 35 to about 55 mol% of SM-102, about 1.5 to about 5 mol% of OH-PEG 2000 -DMG, about 30 to about 40 mol% of cholesterol, and about 5 to about 25 mol% of DSPC. In some embodiments, the lipid component of the nanoparticle composition comprises about 40 to about 55 mol% of SM-102, about 1.5 to about 3 mol% of OH-PEG 2000 -DMG, about 35 to about 40 mol% of cholesterol, and about 10 to about 20 mol% of DSPC. In some embodiments, the lipid component of the nanoparticle composition comprises about 45 to about 50 mol% of SM-102, about 3 to about 5 mol% of OH-PEG 2000 -DMG, about 35 to about 40 mol% of cholesterol, and about 10 to about 20 mol% of DSPC.

[0070] In one or more embodiments, the nanoparticle compositions described herein comprise about 50 mol% of SM-102, about 1.5-3 mol% of OH-PEG 2000- DMG, about 38.5 mole parts of cholesterol, and about 10 mole parts of DSPC.

[0071] The average size of the nanoparticle composition can be between 10 nm and 100 nm, for example, as measured by methods well known in the art. For example, the average size can be about 40 nm to about 150 nm, for example, about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the nanoparticle composition can be about 50 nm to about 120 nm, about 50 nm to about 110 nm, about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, from about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In certain embodiments, the average size of the nanoparticle composition can be about 70 nm to about 110 nm. In particular embodiments, the average size can be about 70 nm.

[0072] The nanoparticle composition can be relatively uniform. A polydispersity index (PDI) can be used to indicate the uniformity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.01 to about 0.10.

[0073] The encapsulation efficiency of a therapeutic and / or prophylactic describes the amount of therapeutic and / or prophylactic that is encapsulated or otherwise associated with the nanoparticle composition after preparation relative to the initial amount provided. Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or prophylactic can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0074] The lipids described herein can be advantageously used in a lipid nanoparticle composition to deliver a therapeutic and / or prophylactic to a local part of a mammal. The lipids described herein have little or no immunogenicity.

[0075] As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means physically connecting the mammalian cell and the nanoparticle. Methods of contacting cells with external entities in vivo and in vitro are well known in the biological arts. For example, a nanoparticle composition can be contacted with a mammalian cell by intravenous injection, and can involve different amounts of the nanoparticle composition. In addition, more than one mammalian cell can be contacted with the nanoparticle composition.

[0076] As used herein, the term "delivering" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic to a subject can involve administering a nanoparticle composition comprising the therapeutic and / or prophylactic to the subject (e.g., by intravenous route). Administering a nanoparticle composition to a mammal or a mammalian cell can involve contacting one or more cells with the nanoparticle composition. "Local delivery" means that the drug is delivered primarily to a partial location of the subject, e.g., to a certain organ, to a certain tissue (e.g., a certain muscle), or to a certain cell (e.g., the organ to which the drug is administered, the muscle to which the drug is administered, the cell to which the drug is administered), and not to organs, tissues, or cells outside of the organ, tissue, or cell. For example, when a drug is used in the liver, the drug is locally delivered to other parts of the liver, and not to organs, tissues, or cells outside of the liver (e.g., through the circulatory system).

[0077] As used herein, the terms "specific delivery," "local delivery" mean that a target cell of interest (e.g., a liver parenchymal cell), tissue or organ (e.g., a mammalian muscle) receives more (e.g., at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a therapeutic and / or prophylactic agent via a nanoparticle as compared to a non-target tissue (e.g., a mammalian liver). The level of nanoparticle delivery to a particular tissue can be identified by comparing the amount of protein produced in a tissue to the weight of the tissue, comparing the amount of therapeutic and / or prophylactic in a tissue to the weight of the tissue, comparing the amount of protein produced in a tissue to the amount of total protein in the tissue, or comparing the amount of therapeutic and / or prophylactic in a tissue to the total amount of therapeutic and / or prophylactic in the tissue. It will be appreciated that the ability of a nanoparticle to be specifically delivered to a target tissue need not be determined in a subject receiving treatment, it can be determined in a surrogate such as an animal model (e.g., a rat model).

[0078] As used herein, "encapsulation efficiency" refers to the amount of a therapeutic and / or prophylactic that becomes part of a nanoparticle composition relative to the initial total amount of the therapeutic and / or prophylactic used to make the nanoparticle composition. For example, if 97 mg of a therapeutic and / or prophylactic out of a total of 100 mg of therapeutic and / or prophylactic initially provided to a composition is encapsulated in the nanoparticle composition, the encapsulation efficiency can be 97%.

[0079] As used herein, "expression" of a nucleic acid sequence includes translation of mRNA into a polypeptide or protein and / or post-translational modification of a polypeptide or protein.

[0080] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., and not within a living organism (e.g., an animal, plant, or microorganism). As used herein, the term "in vivo" refers to events that occur within a living organism (e.g., an animal, plant, or microorganism or a cell or tissue thereof). As used herein, the term "ex vivo" refers to events that occur outside of a living organism (e.g., an animal, plant, or microorganism or a cell or tissue thereof). Ex vivo events can occur in an environment that varies minimally from the natural (e.g., in vivo) environment.

[0081] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. Compounds can include one or more chiral centers and / or double bonds and, therefore, can exist as stereoisomers. The present disclosure encompasses any and all isomers of the compounds described herein, including stereoisomerically pure forms as well as mixtures of enantiomers and stereoisomers, for example, racemic mixtures. Methods for the resolution of enantiomeric and stereoisomeric mixtures and the production of stereoisomerically pure forms are well-known.

[0082] As used herein, a "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, a lipid component can include one or more cationic / ionizable, pegylated, structured, or other lipids, such as a phospholipid.

[0083] As used herein, "administering" refers to other methods of delivering a composition to a subject only intravenously. Targeted delivery (e.g., specific delivery) to a particular region or system of the body can be achieved by intravenous injection.

[0084] As used herein, "modified" means non-natural. For example, an RNA can be a modified RNA. That is, the RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. The RNA also includes any sequence optimization performed on the RNA.

[0085] As used herein, a "nanoparticle composition" is a composition that includes one or more lipids. Nanoparticle compositions are typically on the order of microns or less in size and can include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0086] As used herein, "patient" refers to a subject who can seek or need treatment, who needs treatment, who is undergoing treatment, who will undergo treatment, or who has received treatment by a trained professional for a particular disease or condition.

[0087] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that includes a polyethylene glycol component.

[0088] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0089] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a carrier which can suspend, complex, or dilute the active compound) and has substantially no toxicity and is nontoxic. Excipients can include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and others disclosed herein.

[0090] In this specification, structural formulae of compounds are in some cases presented for the convenience of representing certain isomers, but the disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and the like, it being understood that not all isomers can have the same level of activity.

[0091] For compounds represented by the structural formulae herein, crystalline polymorphism can occur. It is noted that any crystalline form, mixture of crystalline forms, or anhydride or hydrate thereof is included within the scope of the disclosure. The terms "crystalline polymorph," "polymorph," or "crystal form" refer to crystal structures in which a compound (or salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms often have different X-ray diffraction patterns, infrared spectra, melting points, density hardness, crystal shape, optical and electrical properties, stability, and solubility. The dominant crystal form can be influenced by the recrystallization solvent, the rate of crystallization, storage temperature, and other factors. Crystalline polymorphs of a compound can be prepared by crystallization under different conditions.

[0092] The composition can also include a salt of one or more compounds. The salt can be a pharmaceutically acceptable salt. As used herein, “pharmaceutically acceptable salt” refers to a derivative of the disclosed compounds in which the parent compound is altered by the substitution of an existing acid or base moiety with its salt form (e.g., by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral acid salts or organic acid salts of basic residues such as amines; alkali metal salts or organic salts of acidic residues such as carboxylic acids; and the like.

[0093] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like.

[0094] Representative alkali or alkaline earth salts include sodium, lithium, potassium, calcium, magnesium, and the like, and nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0095] Pharmaceutically acceptable salts of the present disclosure include, for example, conventional nontoxic salts of a parent compound formed by the attachment of a nontoxic inorganic or organic acid or base to the parent compound.

[0096] Pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts can be found, e.g., in Remington's Pharmaceutical Sciences, any edition, which is hereby incorporated by reference in its entirety.

[0097] As used herein, a "phospholipid" is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid can include one or more multiple (e.g., double or triple) bonds (e.g., one or more degrees of unsaturation). Particular phospholipids can facilitate fusion with a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). Fusion of a phospholipid with a membrane can allow one or more components of a lipid-containing composition to cross the membrane, thereby allowing, for example, delivery of one or more components to a cell.

[0098] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues typically linked by peptide bonds, which can be produced naturally (e.g., isolated or purified) or synthetically.

[0099] As used herein, "RNA" refers to a ribonucleic acid, which can be naturally or non- naturally occurring. For example, an RNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA can include a cap structure, a chain-terminating nucleoside, a stem loop, a poly A sequence, and / or a polyadenylation signal. An RNA can have a nucleotide sequence that encodes a polypeptide of interest. For example, an RNA can be a messenger RNA (mRNA). Translation of an mRNA that encodes a particular polypeptide, such as in vivo translation of an mRNA within a mammalian cell, can produce the encoded polypeptide. An RNA can be selected from the group consisting of a small interfering RNA (siRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), and an mRNA.

[0100] As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose / one / single route / single point of contact, i.e., a single administration event. As used herein, a "split dose" is a division of a single unit dose or total daily dose into two or more doses. As used herein, a "total daily dose" is an amount given or prescribed to be given in a 24 hour period. It can be administered as a single unit dose.

[0101] As used herein, "size" or "average size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0102] As used herein, the term "subject," "object," or "patient" refers to any organism to which a composition according to the present disclosure can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Preferably, a subject described herein has a disease that would benefit from local delivery of a drug, e.g., a lesion of an organ or tissue to which it is desirable to deliver a drug only to that organ or tissue.

[0103] As used herein, "target cell" refers to any one or more cell(s) of interest. The cell can be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism can be an animal, preferably a mammal, more preferably a human and most preferably a patient.

[0104] As used herein, "target tissue" refers to any one or more tissue type(s) of interest in which delivery of a treatment and / or prophylaxis results in a desired biological and / or pharmacological effect. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. In particular applications, the target tissue can be kidney, lung, spleen, vascular endothelium in a blood vessel (e.g., intracoronary or intrafemoral) or tumor tissue (e.g., by intratumoral injection) or muscle (e.g., by intramuscular injection).

[0105] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject. Therapeutic agents are also referred to as "actives" or "active agents" and include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic drugs, small molecule drugs, proteins, and nucleic acids, such as RNA.

[0106] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent that when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, treats, ameliorates, ameliorates symptoms of, diagnoses, prevents, and / or delays onset of the infection, disease, disorder, and / or condition.

[0107] As used herein, "transfection" refers to the introduction of a substance (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0108] As used herein, the term "treatment" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of a particular infection, disease, disorder, and / or condition. For example, "treating" a cancer can refer to inhibiting survival, growth, and / or spread of a tumor. Treatment can be performed on a subject who does not exhibit symptoms of a disease, disorder, and / or condition and / or on a subject who exhibits only early symptoms of a disease, disorder, and / or condition for the purpose of decreasing risk of developing pathology associated with the disease, disorder, and / or condition.

[0109] Therapeutic and / or prophylactic agents

[0110] The nanoparticle composition can include one or more therapeutic and / or prophylactic agents. The present disclosure is a method of delivering a therapeutic and / or prophylactic agent to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof, comprising administering and / or contacting the mammal with a mammalian cell having a nanoparticle composition comprising a therapeutic and / or prophylactic agent to the mammal.

[0111] The therapeutic and / or prophylactic agent can be a substance that, once delivered to a cell or organ, brings about a desirable change in the cell, organ, or other body tissue or system. Such a substance can be used to treat one or more diseases or disorders. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a particular disease or disorder.

[0112] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide," in its broadest sense, includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi inducing agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and the like.

[0113] In some embodiments, the therapeutic and / or prophylactic agent is an RNA. The RNA that can be used in the compositions and methods described herein can be selected from, but not limited to, short chain, antagonist, antisense strand, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA, small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the mRNA is transcribed from the ATP7B gene. In some embodiments, the coding sequence of the mRNA is set forth in SEQ ID NO: 1.

[0114] Nucleic acids and polynucleotides for use in the present disclosure generally include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region (e.g., a 5-UTR) located 5’-terminally to the first region, a second flanking region (e.g., a 3-UTR) located 3’-terminally to the first region, at least one 5’-cap region, and a 3’-stabilizing region. In some embodiments, the nucleic acid or polynucleotide further includes a poly-A region or a Kozak sequence (e.g., in the 5’-UTR). In some cases, the polynucleotide can include one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, the polynucleotide or nucleic acid (e.g., mRNA) can include a 5’ cap structure, a chain-terminating nucleotide, a stem loop, a polyadenosine sequence, and / or a polyadenylation signal. Any one region of the nucleic acid can include one or more alternative components (e.g., alternative nucleosides).

[0115] The nucleic acids and polynucleotides can include one or more naturally occurring components, including any of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides comprise (a) a 5’-UTR, (b) an open reading frame (ORF), (c) a 3’-UTR, (d) a poly-A tail, and any combination of a, b, c, or d above.

[0116] In some cases, the nucleic acid does not substantially induce an innate immune response of a cell into which the polynucleotide (e.g., mRNA) is introduced.

[0117] Other Components

[0118] In addition to those described in the foregoing sections, the nanoparticle composition can include one or more components. For example, the nanoparticle composition can include one or more hydrophobic small molecules, such as a vitamin (e.g., vitamin A or vitamin E) or a sterol.

[0119] The nanoparticle composition can also include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents, or other components. Permeability enhancer molecules can be, for example, the molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof). Polymers can include those that are biodegradable and / or biocompatible. Polymers can be selected from, but are not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. Surface altering agents can include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytics (e.g., acetylcysteine, nafomine, and erdosteine), and DNases (e.g., rhDNase). Surface altering agents can be disposed within the nanoparticle and / or on the surface of the nanoparticle composition (e.g., by coating, adsorption, covalent attachment, or other processes).

[0120] The nanoparticle composition can also include one or more functionalized lipids. For example, lipids can be functionalized with an alkyne group that can undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. In particular, the lipid bilayer can be functionalized in this manner with one or more groups that can be used to facilitate membrane permeation, cell recognition, or imaging.

[0121] The surface of the nanoparticle composition can also be conjugated to one or more useful antibodies. Functional groups and conjugates for targeted cellular delivery, imaging, and membrane permeation are well known in the art. In addition to these components, the nanoparticle composition can include any material useful in a pharmaceutical composition. For example, the nanoparticle composition can include one or more pharmaceutically acceptable excipients or auxiliary ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid carriers, binders, surface active agents, isotonic agents, thickening agents or emulsifiers, buffers, lubricants, oils, preservatives, and other materials. Excipients such as waxes, butter, colorants, coating agents, flavorings, and fragrances can also be included. Pharmaceutically acceptable excipients are well known in the art (see, e.g., Remington's The Science and Practice of Pharmacy, 21st Edition).

[0122] The nanoparticle compositions can optionally include one or more coatings. For example, the nanoparticle compositions can be formulated in a capsule, film, or tablet having a coating. The capsules, films, or tablets of the compositions described herein can have any useful size, tensile strength, hardness, or density.

[0123] The nanoparticle compositions of the present application are prepared only in a form suitable for the injection route of administration.

[0124] Pharmaceutical compositions

[0125] The nanoparticle compositions can be formulated, in whole or in part, as a pharmaceutical composition. The pharmaceutical composition can include one or more nanoparticle compositions.

[0126] For example, the pharmaceutical composition can include one or more nanoparticle compositions, including one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition can also include one or more pharmaceutically acceptable excipients, such as those described herein. General guidelines for formulating and manufacturing pharmaceutical compositions and medicaments can be found in Remington's The Science and Practice of Pharmacy, 21sted. Conventional excipients and auxiliary ingredients can be used in any of the pharmaceutical compositions, unless the excipient or auxiliary ingredient is incompatible with one or more components of the nanoparticle composition. The amount of excipient in the pharmaceutical composition can be determined by one of skill in the art as needed.

[0127] In some embodiments, the mass ratio of the nanoparticle composition to the therapeutic and / or prophylactic agent in the pharmaceutical composition of the present application is 10-50: 1 or 20-25: 1 (e.g., 10-45: 1, 10-40: 1, 10-35: 1, 10-30: 1, 10-25: 1, 12-25: 1, 15-30: 1, 15-25: 1).

[0128] The relative amounts of the one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary according to the nature of the nanoparticle, the size, and / or the condition of the subject being treated, and further depending on the route of administration of the composition. For example, the pharmaceutical composition can include 0.1% to 100% (wt / wt) of the one or more nanoparticle compositions.

[0129] In certain embodiments, the nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at a temperature of 4 °C or less, e.g., a temperature of between about -150 °C) and between about 0 °C or about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C, or -150 °C). For example, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein can be stable for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months, e.g., at a temperature of 4 °C or less (e.g., between about 4 °C and -20 °C). In one embodiment, the formulation is stable for at least 4 weeks at 4 °C. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise

[0130] The nanoparticle compositions and pharmaceutically acceptable excipients disclosed herein are selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pharmaceutical compositions of the present disclosure have a pH value of between about 7 and 8 (e.g., between 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0).

[0131] In the context of the present disclosure, “stability” and “stable” refer to the nanoparticle compositions and / or pharmaceutical compositions disclosed herein being resistant to chemical or physical changes (e.g., degradation, changes in particle size, aggregation, changes in encapsulation, etc.) under given manufacturing, preparation, transport, storage, and / or use conditions, e.g., when stresses such as shear forces, freeze / thaw stresses, etc. are applied.

[0132] The nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including those patients or subjects, cells, tissues, organs, or systems, or groups thereof, that can benefit from the therapeutic effects provided by the delivery of one or more specific pharmaceutical therapeutic and / or prophylactic agents.

[0133] In the context of the present disclosure, “stability” and “stable” refer to the nanoparticle compositions and / or pharmaceutical compositions disclosed herein being resistant to chemical or physical changes (e.g., degradation, changes in particle size, aggregation, changes in encapsulation, etc.) under given manufacturing, preparation, transport, storage, and / or use conditions, e.g., when stresses such as shear forces, freeze / thaw stresses, etc. are applied.

[0134] The nanoparticle composition and / or pharmaceutical composition comprising one or more nanoparticle compositions can be administered to any patient or subject, including those patients or subjects cells, tissues, organs, or systems, or groups thereof, that can benefit from the therapeutic effects provided by administration of one or more specific drug delivery therapeutic and / or prophylactic agents. In some embodiments, the patient or subject has a disorder of copper metabolism, in some embodiments, the patient or subject has Wilson's disease.

[0135] While "composition" relates primarily to compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to any other mammal.

[0136] It is well understood, and readily apparent to those skilled in the art, that modifications suitable for use in adapting the compositions for administration to various animals are within the scope of the disclosure. Such modifications are well known and can be made by those skilled in ordinary veterinary pharmacology, with only ordinary experimentation needed, if any. Intended subjects for administration of the compositions include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals, such as cows, pigs, hoses, sheep, cats, dogs, mice, and / or rats.

[0137] Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any of the methods known or hereafter developed in the art of pharmacology. In general, such preparative methods include the step of bringing the active ingredient into association with the excipient and / or one or more other accessory ingredients, and then, if necessary or desirable, dividing, shaping, and / or packaging the product into the desired single- or multi-dose unit.

[0138] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as single unit dosages, and / or as multiple unit dosages. As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition containing a predetermined quantity of the active ingredient (e.g., nanoparticle composition(s)). The quantity of the active ingredient is generally equal to the dosage of the active ingredient which would be administered at one time to a subject and / or a fraction of such a dosage. For example, the quantity of the active ingredient can be equal to one day's treatment, one week's treatment, or one month's treatment.

[0139] Pharmaceutical compositions are prepared only in forms suitable for the route and method of administration chosen, e.g., in injectable form. An injectable formulation, e.g., a sterile injectable aqueous or oleaginous suspension, can be formulated according to known techniques using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable formulations can be a sterile injectable solution, suspension, and / or emulsion in a nontoxic parenterally acceptable diluent and / or solvent. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Sterile oil can be used as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. The injectable formulations can be sterilized, e.g., by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

[0140] Suitable devices for delivering the intradermal pharmaceutical compositions described herein include any type of liquid or solid injection device, such as a conventional syringe, a fine gauge syringe, a short needle syringe, a liquid jet injection syringe, a compressed gas accelerated powder injection syringe, a ballistic powder delivery device, and the like.

[0141] Methods and uses

[0142] The present disclosure provides methods of producing a polypeptide of interest in a mammalian cell. The method of producing a polypeptide involves contacting the cell with a nanoparticle composition comprising an mRNA encoding the polypeptide of interest. When the cell is contacted with the nanoparticle composition, the mRNA can be taken up and translated in the cell to produce the polypeptide of interest. In some embodiments, the mRNA is transcribed from an ATP7B gene. In some embodiments, the coding sequence of the mRNA is set forth in SEQ ID NO: 1.

[0143] Generally, the step of contacting a mammalian cell with a nanoparticle composition comprising mRNA encoding a polypeptide of interest can be performed in vivo, ex vivo, in culture, or in vitro. The amount of the nanoparticle composition and / or the amount of mRNA therein that is contacted with the cell can depend on the type of cell or tissue contacted, the mode of administration, the physiochemical characteristics of the nanoparticle composition and mRNA (e.g., size, charge, and chemical composition), and other factors. Indicators of efficiency can include polypeptide translation (as indicated by polypeptide expression), levels of mRNA degradation, and indicators of immune response. In some embodiments, the amount of the nanoparticle composition and the mass of mRNA therein is in a ratio of 10-50: 1 or 20-25: 1 (e.g., 10-45: 1, 10-40: 1, 10-35: 1, 10-30: 1, 10-25: 1, 12-25: 1, 15-30: 1, 15-25: 1). The step of contacting a nanoparticle composition comprising mRNA with a cell can involve or result in transfection. The phospholipids included in the lipid component of the nanoparticle composition can facilitate transfection and / or increase transfection efficiency, e.g., by interacting with and / or fusing to cell membranes or intracellular membranes. Transfection can allow for translation of the mRNA within the cell.

[0144] In some embodiments, the nanoparticle compositions described herein can be used for therapy. For example, the mRNA included in the nanoparticle composition can encode a therapeutic polypeptide (e.g., in a translatable region) and produce the therapeutic polypeptide upon contact and / or entry (e.g., transfection) into a cell. In other embodiments, the mRNA included in the nanoparticle composition can encode a polypeptide that improves or increases the immunological fitness of a subject.

[0145] In certain embodiments, the mRNA included in the nanoparticle composition can encode a recombinant polypeptide that can replace one or more polypeptides that are substantially absent in the cell contacted with the nanoparticle composition. Alternatively, the recombinant polypeptide produced by translation of the mRNA can antagonize the activity of an endogenous protein in, on the surface of, or secreted from the cell. In another alternative, the recombinant polypeptide produced by translation of the mRNA can indirectly or directly antagonize the activity of a biological moiety in, on the surface of, or secreted from the cell. The antagonized biological moiety can include, but is not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins.

[0146] Methods of delivering therapeutic agents to cells and organs

[0147] The present disclosure provides methods of delivering a therapeutic and / or prophylactic agent to a mammalian cell or organ. Delivery of a therapeutic and / or prophylactic agent to a cell involves administering to a subject a nanoparticle composition comprising the therapeutic and / or prophylactic agent, wherein administration of the composition involves contacting the cell with the composition. For example, a protein, a cytotoxic agent, a radioactive ion, a chemotherapeutic agent, or a nucleic acid (e.g., RNA, e.g., mRNA) can be delivered to a cell or organ. In the case where the therapeutic and / or prophylactic agent is mRNA, when the cell is contacted with the nanoparticle composition, the translatable mRNA can be translated in the cell to produce a polypeptide of interest. However, essentially non-translatable mRNA can also be delivered to a cell.

[0148] In some embodiments, the nanoparticle composition can target a specific type of cell (e.g., a cell of a particular organ or system thereof). For example, a nanoparticle composition comprising a therapeutic and / or prophylactic agent of interest can be specifically delivered to a mammalian liver, kidney, spleen, femur, or lung with substantially no delivery to other types of cells. Specific delivery to a particular type of cell, organ, or system or tissue thereof means that a higher proportion of the nanoparticle composition comprising the therapeutic and / or prophylactic agent is delivered to the tissue of interest (e.g., tissue) relative to other destinations. In some embodiments, the tissue of interest is selected from the group consisting of liver, kidney, lung, spleen, femur, muscle, tumor tissue (e.g., by intratumoral injection).

[0149] As another example of targeted or specific delivery, mRNA encoding a protein binding ligand (e.g., an antibody or functional fragment thereof, a scaffold protein, or a peptide) or receptor on the surface of a cell can be included in the nanoparticle composition. In some embodiments, the ligand can be a surface-bound antibody, which can allow for modulation of cell targeting specificity. These methods can improve the affinity and specificity of the targeted interaction. The ligand can be selected by, for example, a person of skill in the biological arts based on the desired localization or function of the cell. Targeted cells can include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiomyocytes, adipocytes, vascular smooth muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, white blood cells, granulocytes, and tumor cells. In some embodiments, the targeted cell is a liver parenchymal cell.

[0150] Methods of treating diseases and disorders

[0151] Nanoparticle compositions can be used to treat a disease or disorder. In particular, such compositions can be used to treat a disease or disorder characterized by a loss or abnormality of a protein or polypeptide activity. For example, a nanoparticle composition comprising an mRNA encoding a missing or abnormal polypeptide can be administered or delivered to a cell. Subsequent translation of the mRNA can produce the polypeptide, thereby reducing or eliminating problems caused by the loss or abnormal activity of the polypeptide. The methods and compositions can be used to treat acute diseases or disorders, such as sepsis, stroke, and myocardial infarction. Therapeutic and / or prophylactic agents included in the nanoparticle compositions can also be able to alter the rate of transcription in a given species, thereby affecting gene expression. Diseases and / or disorders characterized by a dysfunctional or abnormal protein or polypeptide activity that can be administered the compositions include, but are not limited to, rare diseases, infectious diseases (as vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0152] In some embodiments, the pharmaceutical compositions described herein can be used to treat a related disease or disorder that targets or biomarker ATP7B. In some embodiments, the related disease or disorder that targets or biomarker ATP7B includes Wilson’s disease.

[0153] The present disclosure provides methods involving administration of a nanoparticle composition comprising one or more therapeutic and / or prophylactic agents and pharmaceutical compositions comprising the same. The terms therapeutic and prophylactic are used interchangeably herein. Therapeutic compositions or imaging, diagnostic, or prophylactic compositions thereof can be administered to a subject using an effective prophylactic, therapeutic, diagnostic, or imaging amount and any route of administration. The specific amount administered to a subject can vary depending on the species, age, and general condition of the subject, the particular ingredients, and the mode of administration. Compositions according to the present disclosure can be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the specific amount of the compositions of the present disclosure to be administered will be decided by the attending physician within the scope of sound medical judgment. Nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents are administered by intravenous injection only.

[0154] In certain embodiments, the compositions according to the present disclosure can be administered at a level sufficient to deliver a dose of about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 2.5 mg / kg mg / kg, about 0.001 mg / kg to about 2.5 mg / kg, about 0.005 mg / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, about 0.05 mg / kg to about 0.05 mg / kg to about 2.5 mg / kg about 2.5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, about 1 mg / kg to about 2.5 mg / kg, about 0.0001 mg / kg to about 1 mg / kg, about 0.001 mg / kg to about 1 mg / kg, about 0.005 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg of a therapeutic and / or prophylactic agent (e.g., mRNA).

[0155] Doses can be administered in the same or different amounts one or more times per day to achieve the desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dose can be delivered, for example, three times per day, twice per day, once per day, every other day, every third day, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations) can be used to deliver the desired dose.

[0156] Nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. Each agent will be administered at a dose and / or schedule determined for that agent. Therapeutic, prophylactic, diagnostic, or imaging agents used in combination can be administered together in a single composition or separately in different compositions.

[0157] In some embodiments, the present application also provides a method of treating hepatolenticular degeneration, comprising providing to a subject suffering from hepatolenticular degeneration a pharmaceutical composition as described in any of the embodiments herein.

[0158] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the disclosure is not intended to be limited to the above description but is to be accorded the full scope inherent to the principles described herein.

[0159] In the claims, such as in the claims recited below, the articles "a," "an," and "the" can mean one or more than one unless otherwise indicated or where the use can refer to a singular sense that is clear from the context.

[0160] The term "comprising" is intended to be open and permits but does not require including additional elements or steps. Where the term "comprising" is used, the terms "consisting essentially of and "consisting of are also thereby encompassed and disclosed. Further, it is to be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Two or more steps or actions can be conducted simultaneously.

[0161] The present application has the following beneficial effects:

[0162] The present application constructs a liver parenchymal cell targeted nucleic acid delivery vector expressing a protein of interest, and after encapsulating the delivery vector with EGFP mRNA, the mouse is injected via tail vein. The results show that the delivery vector can significantly improve the expression of EGFP protein in liver parenchymal cells and reduce the expression of EGFP protein in liver non-parenchymal cells. Using a mouse model of hepatolenticular degeneration, we verified the liver copper excretion function of the ATP7B mRNA delivery vector. ICP-MS results show that the liver copper concentration of the ATP7B mRNA / LNP treatment group is significantly reduced, and H&E results also demonstrate that ATP7B mRNA / LNP treatment can reduce liver lesions.

[0163] The present application will be further described in the following with specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present application. The methods and reagents used in the examples are conventional methods and reagents in the art, unless otherwise specified.

[0164] Experimental materials

[0165] Experimental methods

[0166] 1. LNP preparation

[0167] The four components of the organic phase lipid were dissolved in anhydrous ethanol to 10 mg / mL, and prepared according to the molar ratio of the following table prescription; 1 mg / mL of Luc (EGFP mRNA) was prepared into 70 μg / mL as the aqueous phase with 25 mM sodium acetate buffer solution; the two-phase channel of the microfluidic instrument (PDMS chip) was cleaned with anhydrous ethanol and pure water respectively, and then the aqueous phase was rinsed twice with 25 mM sodium acetate buffer solution. At the beginning of preparation, the two-phase pipeline was first used to discharge 300 μL of air in the pipeline with the prepared organic phase component and aqueous phase component respectively, and then 1:3 of the preparation solution of the organic phase and the aqueous phase was taken, and the LNP was prepared at a flow rate of 4 mL / min and 12 mL / min of the organic phase and the aqueous phase respectively, and the first 0.5 mL of effluent was discarded, and only the subsequent effluent was collected until the end.

[0168] Molar ratio of components in the preparation

[0169] 1.1 LNP ultrafiltration

[0170] The prepared LNP stock solution was added to 5 times the volume of PBS, and an ultrafiltration centrifuge tube with an inner tube volume of 15 mL and a molecular weight cutoff of 100 KDa was used at 2000-3000 rpm for 10 min to obtain an ultrafiltrate volume of about 1 mL. 10 times the volume of PBS was added to the filtrate to wash the ultrafiltration tube filter membrane again, and centrifugation was performed to obtain a filtrate volume of about 1 mL. 10 times the volume of PBS was added to the filtrate to wash the ultrafiltration tube filter membrane again, and centrifugation was performed to obtain a filtrate volume of about 1 mL. After repeatedly washing the filter membrane, centrifugation was performed to obtain a final 1 mL of filtrate which was placed in an enzyme-free EP tube and stored in a 4°C refrigerator for standby.

[0171] 2. Content and encapsulation efficiency determination

[0172] 2.1 Standard curve preparation

[0173] The mRNA stock solution was diluted with 1×TE to 2 μg / mL, and 0, 2, 10, 25, 50, and 100 μL of each concentration standard solution was taken into a 96-well black enzyme-labeled plate, and then the operation was performed according to the RNA quantification kit label (1×TE, 100 μL, 1×Ribo, 100 μL). The enzyme-labeled instrument was detected under the condition of excitation wavelength 480 nm and emission wavelength 520 nm.

[0174] 2.2 LNP measurement sample preparation

[0175] Take the ultrafiltration of LNP first with 1xTE dilution to the theoretical concentration of about 1 μg / mL; Permeation: diluted LNP with a final concentration of 1% Triton X-100 room temperature permeation 10 min; Non-permeation: diluted LNP with equal volume of DEPC water at room temperature for 10 min. Then take 100 μL of each sample to 96-well black enzyme plate, and add 1xRibo 100 μL, 3 replicates for each sample, and detect under the condition of excitation wavelength 480 nm and emission wavelength 520 nm on the microplate reader.

[0176] 3. Particle size determination

[0177] Take the ultrafiltration of LNP, dilute 10 times with pure water, and then add it to the particle size pool. Measure the particle size on the Malven particle size instrument.

[0178] 4. Method of each embodiment

[0179] 4.1 EGFP protein expression in liver parenchymal cells

[0180] Select 8-week-old male C57 mice, weighing about 20 g each. Use a fine syringe to inject 100 μL of 1.5% -OH LNP, 3% -OH LNP and 5% -OH LNP (1 mg / kg, pH 7.4, 20 μg EGFP mRNA per mouse, 3 mice per group, diluent is PBS) into the tail vein. At 8h and 24h after administration, isoflurane anesthesia is performed, the peritoneum is cut open, the inferior vena cava and portal vein are exposed, and HBSS-0.5mM EGTA and type IV collagenase solution (100U / mL) are perfused in turn at the inferior vena cava. After perfusion, the liver is peeled off and cut into small pieces, 5 mL of type IV collagenase solution is added, and the liver tissue is incubated in a 37°C water bath for 0.5h (90rpm) for digestion; After incubation, pass through a 70 μm cell sieve, and wash with 15 mL of cold HBSS-CaCl2 (pre-cooled in a 4°C refrigerator), collect the filtrate, and centrifuge (4°C, 50g, 3min); Upper layer: liver parenchymal cells, lower layer: liver non-parenchymal cells; Select cell-specific markers to separate liver parenchymal cells, liver Kupffer cells (liver macrophages), liver B cells, liver T cells, and analyze EGFP protein expression in each type of cell. The tail vein injection of 100 μL of PBS group is used as a negative control.

[0181] 4.2 ATP7B cell expression (Huh 7)

[0182] Take the Huh7 cells in the logarithmic phase, use trypsin to digest the cells, count the cells, resuspend to 2.5x10^5 / ml. Add 2ml of resuspended cell suspension (5x10^5 cells per well) to a 6-well plate, shake evenly to cover the bottom of the plate, and return to the incubator for 14-16h to make it completely adherent. Transfect ATP7B mRNA into Huh7 cells using LNP. The transfection amount is 1 or 3 μg / well. Remove the LNP-containing medium after 4h, and replace it with fresh medium. Remove the medium after 24h, and rinse with 2ml of PBS. Then add 200μL of 1X loading buffer to the plate, shake the bottom of the dish to lyse the cells and take out to 1.5mL of enzyme-free EP tube, heat in boiling water bath for 10min. Take 10μL of the above sample and add it to a 4-20% precast gel, run at 80V until the bromophenol blue reaches the bottom of the gel plate to stop electrophoresis, and turn off the power. After electrophoresis, remove the gel from the gel plate and transfer it to the Trans-Blot Turbo Transfer Stater System according to the instrument instructions; complete the membrane transfer according to the instrument instructions. Take out the PVDF membrane, place it with the front side facing up in the incubation tank, and add blocking solution (5% BSA) at room temperature for 1h. Remove the blocking solution, add the primary antibody according to the antibody dilution ratio in the experimental materials, and incubate overnight at 4°C. Remove the primary antibody, add 1x TBST until the PVDF membrane is completely covered, place the incubation tank on the shaker at 90rpm and wash for 10min, repeat 3 times. Remove 1x TBST, add the corresponding secondary antibody according to the antibody dilution ratio in the experimental materials, and incubate the incubation tank on the shaker at 60rpm at room temperature for 1h. Remove the secondary antibody, add 1x TBST until the PVDF membrane is completely covered, place the incubation tank on the shaker at 90rpm and wash for 10min, repeat 3 times. Remove 1x TBST, and use ECL luminescent solution to expose and take pictures in the chemiluminescence imaging system.

[0183] 4.3 Verification of cell copper excretion function

[0184] Take the Huh7 cells in the logarithmic phase, use trypsin to digest the cells, count the cells, resuspend to 1x10^5 / ml. Add 0.5ml of resuspended cell suspension (5x10^4 cells per well) to 3 24-well plates, shake evenly to cover the bottom of the plate, and label them as plate 1, plate 2 respectively. Return to the incubator for 14-16h to make it completely adherent. Transfect 0.5μg of ATP7B mRNA into each well of plate 1 using Lipo2000. 2h later, transfect 0.5μg of pGL3-4MRE-LUC plasmid into each well of plate 1 and plate 2 using Lipo2000. Remove the lipo-containing medium after 4h, and replace it with fresh medium.

[0185] Copper-containing medium configuration: Filter-sterilized 50 mmol / L copper sulfate solution was added to complete medium to make the final concentration of copper sulfate 0, 20, 40, 60, 80, 100, 150 μmol / L, a total of 8 concentrations. After 24 h, the medium was removed, and then the copper-containing medium was added to the plate, 3 replicates per plate and per concentration. After 24 h, the copper-containing medium was removed and rinsed with 2 ml of PBS. Then trypsin digestion was used, the cells were collected by centrifugation, resuspended with 50 μL of PBS, and transferred to a white opaque 96-well plate. Add 50 μL of ONE-Glo reagent, shake well, and incubate at room temperature for 10 min. Use a microplate reader to read the luminescence data.

[0186] 4.4 ATP7B expression in hepatolenticular degeneration mice

[0187] Select 8-week-old male hepatolenticular degeneration mice, weighing about 20 g each. Inject 100 μL of 3% -OH LNP (20 μg of ATP7B mRNA per mouse) into the tail vein using a fine syringe. At 8 h after administration, paraffin-embedded mouse livers were taken by perfusion, sectioned, and subjected to immunohistochemistry and immunofluorescence experiments. The tail vein injection of 100 μL of PBS group was used as a negative control.

[0188] 4.5 ATP7B mRNA-LNP reduces copper ion accumulation in the livers of hepatolenticular degeneration mice

[0189] Select 4-6-week-old hepatolenticular degeneration mice, weighing about 20 g each. Inject 100 μL of 3% -OH LNP (40 μg of ATP7B mRNA per mouse, i.e., 2 mg / kg) into the tail vein using a fine syringe, once a week. After two months of administration, part of the mouse liver was taken by perfusion for oven drying and ICP-MS determination of copper ion concentration, and another part of the liver was subjected to H&E staining to observe liver damage. The tail vein injection of 100 μL of PBS group was used as a negative control.

[0190] Example 1: Particle size, PDI, % encapsulation

[0191] The particle size distribution, encapsulation rate, and polydispersity index of LNP carriers with different PEG lipid contents are shown in FIG. 1. The particle size of 1.5% -OH was 104.0 ± 1.801 nm, the PDI was 0.028 ± 0.023, and the encapsulation rate was 91.17%; the particle size of 3% -OH was 89.09 ± 0.7905 nm, the PDI was 0.087 ± 0.029, and the encapsulation rate was 94.84%; and the particle size of 5% -OH was 72.64 ± 1.109 nm, the PDI was 0.082 ± 0.052, and the encapsulation rate was 96.55%. The above LNP formulations have good quality in each parameter, meeting the needs of subsequent experiments.

[0192] Example 2: EGFP protein expression in hepatocytes

[0193] Intravenous injection of 1.5%-OH LNP, 3%-OH LNP and 5%-OH LNP (1 mg / kg, 20 μg EGFP mRNA / each, 3 each group), 8h, 24h after administration, flow cytometry analysis of liver parenchymal cells and non-parenchymal cells EGFP protein expression. Figure 2(A) is the EGFP protein expression of liver parenchymal cells in each experimental group; Figure 2(B) is the EGFP protein expression of liver T cells; Figure 2(C) is the EGFP protein expression of liver B cells; Figure 2(D) is the EGFP protein expression of liver macrophages. Compared with 1.5%-OH LNP and 5%-OH LNP, 3%-OH LNP can improve the EGFP protein expression of liver parenchymal cells, and the effect is most obvious at 8h, and the average fluorescence intensity is about twice that of the other two experimental groups, while reducing the EGFP protein expression of liver B cells and T cells. As can be seen from Figure 2(D), increasing the proportion of PEG lipid modification in LNP (from 1.5% to 3%, 5%) can significantly reduce the EGFP protein expression of liver macrophages. From the above results, the inventors screened a nucleic acid delivery carrier with liver parenchymal cell expression advantage by optimizing the proportion of PEG lipid in LNP prescription.

[0194] Example 3: ATP7B cell expression

[0195] 3% OH ATP7B mRNA / LNP transfection of ATP7B mRNA to Huh7 cells, and the expression effect was investigated by immunoblotting experiment 24h after transfection. As shown in Figure 3, the Huh7 cells transfected with ATP7B mRNA showed corresponding bands at 150kD, proving the successful expression of ATP7B protein.

[0196] Example 4: Verification of ATP7B protein cell copper excretion function

[0197] A copper concentration responsive LUC plasmid was constructed to verify the cell copper excretion function of ATP7B protein. The structure of copper concentration responsive LUC plasmid is shown in Figure 4(A), and the LUC protein expression and intracellular copper ion concentration can be positively correlated by introducing MRE structure. As shown in Figure 4(B), the intracellular copper ion concentration of cells without transfection of ATP7B mRNA increases with the increase of the copper ion concentration of the culture medium; while the increase multiple of the intracellular copper ion concentration of cells transfected with ATP7B mRNA is significantly lower than that of the untransfected group, proving the copper excretion function of ATP7B protein.

[0198] Example 5: Hepatolenticular degeneration mouse ATP7B expression

[0199] Male hepatolenticular degeneration mice were injected with 3% -OH ATP7B mRNA / LNP (1 mg / kg, 2 mg / kg) via tail vein. The in vivo expression performance was investigated by immunoblotting experiment, liver tissue immunoproteomics section and immunofluorescence section. Figure 5(A) is the immunoblotting experiment result of liver tissue of different experimental groups, Figure 5(B) is the immunoproteomics section of liver tissue of different experimental groups, and Figure 5(C) is the immunofluorescence section of liver tissue of different experimental groups. The immunoblotting map shows the successful expression of ATP7B protein in mice at 150kD. The immunohistochemical section result shows the extensive expression of ATP7B protein in mouse liver tissue. Figure 5(C) shows DAPI in blue, which shows the nucleus of the cell, and ATP7B in red, which also proves the extensive expression of ATP7B protein in mouse liver tissue.

[0200] Example 6: ATP7B mRNA-LNP reduces copper ion accumulation in the liver of hepatolenticular degeneration mice

[0201] 4-6 week-old hepatolenticular degeneration mice were selected and injected with 100 μL of 3% -OH LNP (40 μg of ATP7B mRNA per mouse, i.e. 2 mg / kg) via tail vein, once a week. After two months of administration, part of the mouse liver was taken for ICP-MS determination of copper ion concentration after drying, and another part of the liver was subjected to H&E staining to observe the liver damage. As shown in Figure 6(A), obvious necrosis of liver tissue was observed in the PBS group, with small and obviously aggregated nuclei, and no such phenomenon was observed in the WD (2mpk) group and the WT (normal mouse) group. Figure 6(B) shows the copper content of the liver tissue of mice in each experimental group. Compared with the WD (PBS) group, the WD (2mpk) group significantly reduced the liver copper content from 400 μg / g to 200 μg / g. At the same time, the urine copper content of the WD (2mpk) group was also significantly reduced, indicating that after the expression of ATP7B protein in the liver, the copper pathway metabolized by the kidney was weakened, which could reduce the metabolic burden of the kidney.

[0202] Part of the sequence in this paper

[0203] SEQ ID NO: 1 CDS sequence of ATP7B mRNA

Claims

1. A nanoparticle composition, characterized in that, The nanoparticle composition comprises a cationic lipid, a PEG lipid, a phospholipid, and a structural lipid; wherein the lipid component of the PEG lipid is 1.0-5.5 mol%; Preferably, the nanoparticle composition further comprises a therapeutic agent and / or a prophylactic agent.

2. The nanoparticle composition of claim 1, wherein, The nanoparticle composition has one or more of the following characteristics: The cationic lipid is selected from one or more of DLin-KC2-DMA, D-Lin-MC3-DMA, DOTMA, DOTAP, DODAP, DOP-DEDA, DODMA, DC-Chol, D-Lin-MC3-DMA, and SM-102; PEG-modified phosphatidyl ethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkyl amine, PEG-modified diacyl glycerol, PEG-modified dialkyl glycerol, and mixtures thereof; preferably, the PEG-lipid is selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids; preferably, the lipid is selected from PEG-DMG, OH-PEG-DMG, PEG-DSPE, PEG-DSG, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-DAG, PEG-DPPE, PEG-c-DMA, or DMG-PEG 2000 ; more preferably, the PEG-lipid is OH-PEG 2000 -DMG; the phospholipid is a lipid of (I); wherein Rp represents a phospholipid moiety, R1 and R2 represent a fatty acid moiety with or without unsaturation, which can be the same or different; the phospholipid moiety can be selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin; the fatty acid moiety can be selected from lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, a-linolenic acid, erucic acid, phytanic acid, eicosanoic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid; Preferably, the phospholipid is selected from DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, DUPC, POPC, OChemsPC, DOPG, and sphingomyelin; and The structural lipid is selected from one or more of Cholesterol, Dehydrocholesterol, Dihydrocholesterol, Desmosterol, Coprostanol, Sitosterol, β-Sitosterol, Ergocalciferol, Dihydroergocalciferol, Cholestanol, Ergosterol, Dihydroergosterol, Melitoasterol, Epicholesterol, Fucosterol, Hexahydrofurosterol, Hydroxycholesterol, Campesterol, Stigmasterol, Stigmastanol, Tomato base, Tomato base, Cholic acid, Ursolic acid, α-Tocopherol, Glycocholic acid, Taurocholic acid, Deoxycholic acid, and Lithocholic acid; preferably, the structural lipid is Cholesterol; preferably, the Cholesterol is a polypeptide-modified Cholesterol; preferably, the structural lipid comprises Cholesterol and a corticosteroid or a combination thereof.

3. A pharmaceutical composition comprising the nanoparticle composition of claim 1 or 2 and a pharmaceutically acceptable excipient, which is a locally acting pharmaceutical composition; Preferably, the therapeutic agent and / or prophylactic agent is RNA; Preferably, the RNA is selected from short chain, antagonist, antisense, ribozyme, small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, transfer RNA, messenger RNA, and a mixture thereof; Preferably, the mRNA is transcribed from an ATP7B gene; more preferably, the coding sequence of the mRNA is shown in SEQ ID NO:

1.

4. A method of delivering a therapeutic and / or prophylactic agent to a liver cell, the method comprising the step of administering to a subject a nanoparticle composition, the composition comprising: (1) a lipid component comprising a cationic lipid, a phospholipid, a PEG lipid, and a structural lipid, and (2) a therapeutic and / or prophylactic agent, wherein the administration comprises contacting a liver cell with the nanoparticle composition, thereby delivering the therapeutic and / or prophylactic agent to the liver cell; wherein the lipid component of the PEG lipid is 1.0-5.5 mole %.

5. The method of claim 4, wherein, the lipid component of the PEG lipid is 1.0 mole % to 5.0 mole %; preferably, the lipid component of the PEG lipid is 1.5 mole % to 5.0 mole %; preferably, the lipid component of the PEG lipid is 1.5 mole % to 3.0 mole %; preferably, the lipid component of the PEG lipid is 3.0 mole % to 5.0 mole %; preferably, the lipid component of the PEG lipid is 2.0 mole % to 4.5 mole %; preferably, the subject is a mammal, e.g., a human; preferably, the liver cell is a hepatocyte.

6. A method of producing a polypeptide in a hepatocyte, the method comprising the step of contacting a hepatocyte with a nanoparticle composition, the composition comprising: (1) a lipid component comprising a cationic lipid, a phospholipid, a PEG lipid, and a structural lipid, and (2) an mRNA encoding the polypeptide, from which mRNA the polypeptide is translatable in a liver cell to produce a polypeptide; preferably, the liver cell is a hepatocyte.

7. A method of treating a disease or condition in a mammal, or effecting a therapeutic and / or prophylactic agent locally, by administering the therapeutic and / or prophylactic agent to hepatocytes, the method comprising the step of administering to the hepatocytes of the mammal a therapeutically effective amount of a nanoparticle composition, the nanoparticle composition comprising: (1) a lipid component comprising a cationic lipid, a phospholipid, a PEG lipid, and a structural lipid, and (2) a therapeutic and / or prophylactic agent.

8. The method of claim 7, wherein, The method has one or more of the following features: the disease or condition is a disease or condition of a dysfunctional or abnormal protein or polypeptide; the disease is a disease that benefits from delivery of a therapeutic and / or prophylactic agent into a liver cell; the disease or condition is selected from the group consisting of rare diseases, infectious diseases, cancers and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases; the liver cell is a hepatocyte; and / or the disease or condition is Wilson's disease.

9. Use of the nanoparticle composition of claim 1 or 2 and / or the pharmaceutical composition of claim 3 in the manufacture of a medicament for the treatment or prevention of a disease; preferably, the disease is a disease that benefits from delivery of a therapeutic and / or prophylactic agent into a hepatocyte; more preferably, the disease is Wilson's disease.

10. A method of producing the nanoparticulate matter of claim 1 or 2, characterized in that, The method comprises mixing the components of the nanoparticle composition. the lipid component of the PEG lipid is 1.0 mole % to 5.0 mole %; preferably, the lipid component of the PEG lipid is 1.5 mole % to 5.0 mole %; preferably, the lipid component of the PEG lipid is 1.5 mole % to 3.0 mole %; preferably, the lipid component of the PEG lipid is 3.0 mole % to 5.0 mole %; preferably, the lipid component of the PEG lipid is 2.0 mole % to 4.5 mole %; preferably, the subject is a mammal, e.g., a human; preferably, the liver cell is a hepatocyte. (1) a lipid component comprising a cationic lipid, a phospholipid, a PEG lipid, and a structural lipid, and (2) an mRNA encoding the polypeptide, from which mRNA the polypeptide is translatable in a liver cell to produce a polypeptide; preferably, the liver cell is a hepatocyte. (1) a lipid component comprising a cationic lipid, a phospholipid, a PEG lipid, and a structural lipid, and (2) a therapeutic and / or prophylactic agent. The method has one or more of the following features: the disease or condition is a disease or condition of a dysfunctional or abnormal protein or polypeptide; the disease is a disease that benefits from delivery of a therapeutic and / or prophylactic agent into a liver cell; the disease or condition is selected from the group consisting of rare diseases, infectious diseases, cancers and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases; the liver cell is a hepatocyte; and / or the disease or condition is Wilson's disease.

9. Use of the nanoparticle composition of claim 1 or 2 and / or the pharmaceutical composition of claim 3 in the manufacture of a medicament for the treatment or prevention of a disease; preferably, the disease is a disease that benefits from delivery of a therapeutic and / or prophylactic agent into a hepatocyte; more preferably, the disease is Wilson's disease. The method comprises mixing the components of the nanoparticle composition.

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