An mRNA composition and uses thereof

Abstract

The present disclosure belongs to the technical field of biological medicine, and relates to an mRNA composition and application thereof, wherein the mRNA composition comprises a lipid nanoparticle and mRNA contained in the interior of the lipid nanoparticle, and the mRNA can express CXCL3. After the mRNA composition of the present disclosure is infused through the tail vein, a large number of monocyte-derived macrophages can be chemotactically infiltrated into the cirrhotic liver of a mouse, and the macrophages can phagocytize fibrosis, thereby significantly reducing the fibrosis level in the liver of the mouse. Experimental results show that the composition has almost no toxicity to normal tissues, has high safety, and shows potential in treating liver fibrosis.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biomedicine, in particular to an mRNA-lipid nanoparticle complex and a preparation method and application thereof. BACKGROUND

[0002] Under the background of chronic liver injury such as long-term viral infection, alcoholism, high-fat diet, autoimmune disease, etc., liver fibrosis is formed by excessive deposition of extracellular matrix (ECM). Liver fibrosis is a necessary pathway for various chronic liver diseases, and will develop into cirrhosis and even liver cancer if it continues to progress. Inhibiting or even reversing liver fibrosis and cirrhosis has always been a hot spot in the field of liver disease research, but so far no significant progress has been made. Current treatment strategies for liver fibrosis can only delay the progression of fibrosis, but cannot promote ECM degradation to reverse fibrosis. Therefore, it is urgent to explore new treatment methods for liver fibrosis.

[0003] The number of liver macrophages is huge. A large number of basic experimental studies have shown that macrophages play a crucial role in the reversal of endogenous liver fibrosis. However, the cell therapy program of direct infusion of macrophages usually has the disadvantages of long cycle, strict process requirements, high treatment cost, etc., and is difficult to be applied and popularized.

[0004] mRNA therapy has become a research hotspot in the field of drug development. mRNA produces corresponding proteins through translation to exert biological functions. In theory, mRNA can replace drugs on the protein level. mRNA therapy is particularly suitable for treating diseases that do not require continuous and lifelong expression of therapeutic proteins, and has the advantages of easy scale-up production and no potential risk of insertion mutation. SUMMARY

[0005] One of the purposes of the present disclosure is to provide an mRNA-lipid nanoparticle complex for treating cirrhosis in a safe and effective manner by promoting macrophage infiltration and degrading fibrosis.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present disclosure is as follows:

[0007] In a first aspect, the present disclosure provides an mRNA composition comprising mRNA and a lipid nanoparticle, wherein the mRNA is contained in the lipid nanoparticle, and the mRNA is capable of expressing chemokine ligand 3 (CXCL3).

[0008] In some embodiments, the amino acid sequence of the CXCl3 is set forth in SEQ ID NO: 2. In some embodiments, the mRNA comprises a CXCL3 coding region comprising the nucleotide sequence set forth in SEQ ID NO: 5 or comprising a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 5.

[0009] In some embodiments, the nucleotide sequence of the CXCL3 coding region is set forth in SEQ ID NO: 5.

[0010] In some embodiments, the mRNA further comprises a 5’ cap structure, a 5’ UTR, a Kozak sequence, a 3’ UTR, and / or a poly-A region.

[0011] In some embodiments, the 5’ UTR sequence is the sequence set forth in SEQ ID NO: 6.

[0012] In some embodiments, the 3’ UTR sequence is the sequence set forth in SEQ ID NO: 8.

[0013] In some embodiments, the mRNA comprises the nucleotide sequence set forth in SEQ ID NO: 4 or comprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 4; in some preferred embodiments, the nucleotide sequence of the mRNA is set forth in SEQ ID NO: 4.

[0014] In some embodiments, the lipid nanoparticle comprises an ionizable lipid, a non-cationic lipid, cholesterol, and a polyethylene glycol (PEG) lipid.

[0015] In some embodiments, the ionizable lipid is selected from (4-hydroxybutyl) aminobis(hexane-6,1 -diiyl) bis(2-hexyldecanoate) (ALC-0315, CAS No.: 2036272-55-4), 1,1 '-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1 -yl)ethyl)amino)bis(dodecan-2-ol) (C12-200), 4-(N,N-dimethylamino)butanoic acid-6,9,28,31 -tetraen-19-triacontanol ester (MC3), 1,2-dioleoyl-N,N-dimethyl-3-aminopropane (DLinDMA), 2,2-dioleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2DMA), (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1 H-cyclopenta[a]phenanthren-3-yl 3-(1 H-imidazol-4-yl)propanoate ethyl ester (ICE), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1 -yl) tetracos-15,18-dien-1 -amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1 -yl) tetracos-4,15,18-triethylenetetraamine-1 -amine (HGT5001 ), 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1 -yl]amino]butyl]-2,5-piperazinedione (OF-02), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1 -(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 5-carboxyspermyl glycine-dioctadecanamide (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1 -(cis,cis-9,12-octadecenyl)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β- yloxy)-3'-oxapentyl]-3-dimethyl-1-(cis,cis-9',1-2'-octadecenyl)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'- dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyl- oxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamoyl-3- dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamoyl (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminooxylethyl-[1,3]-dioxolane (DLin-K- XTC2-DMA), 2-((2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)- N,N-dimethylethanamine (HGT4003). In some preferred embodiments, the ionizable lipid is ALC-0315.

[0016] In some embodiments, the non-cationic lipid is selected from one or more of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)- cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2- oleoylphosphatidyethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (trans DOPE). In some preferred embodiments, the non-cationic lipid is distearoylphosphatidylcholine.

[0017] In some embodiments, the PEG lipid is selected from one or several of PEG-phospholipids and PEG-ceramides, for example from one or several of methoxypolyethylene glycol bimyristyl acetamide (ALC-0159), dimyristylglycerol-PEG2000 (DMG-PEG2000), distearoylphosphatidylethanolamine-PEG2000 (DSPE-PEG2000), dipalmitoylphosphatidylethanolamine-PEG2000 (DPPE-PEG2000), distearoylphosphatidylethanolamine-PEG2000 (DMPE-PEG2000), dioleoylphosphatidylethanolamine-PEG2000 (DOPE-PEG2000), distearoylphosphatidylethanolamine-PEG1000 (DSPE-PEG1000), dipalmitoylphosphatidylethanolamine-PEG1000 (DPPE-PEG1000), dimyristoylphosphatidylethanolamine-PEG1000 (DMPE-PEG1000), dioleoylphosphatidylethanolamine-PEG1000 (DOPE-PEG1000), distearoylphosphatidylethanolamine-PEG550 (DSPE-PEG550), dipalmitoylphosphatidylethanolamine-PEG550 (DPPE-PEG550), dimyristoylphosphatidylethanolamine-PEG550 (DMPE-PEG550), PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, PEG550-ceramide. In some embodiments, the PEG lipid is ALC-0159.

[0018] In some preferred embodiments, the lipid nanoparticle comprises the ionizable lipid ALC-0315, distearoylphosphatidylcholine, cholesterol, and the PEG lipid ALC-0159.

[0019] In some embodiments, the lipid nanoparticle has a particle size of 85-120 nm, for example can be 85 nm, 86 nm, 87 nm, 88 nm, 90 nm, 92 nm, 94 nm, 96 nm, 98 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, preferably 88-89 nm, more preferably 88 nm.

[0020] In some embodiments, the monodispersity of the lipid nanoparticle is 0.030-0.99, for example, can be 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.063, 0.065, 0.067, 0.070, 0.073, 0.075, 0.077, 0.080, 0.083, 0.085, 0.087, 0.090, 0.095, 0.099, preferably 0.065-0.085, more preferably 0.075.

[0021] In some embodiments, the zeta potential of the lipid nanoparticle is 3.0-8.0 mV, for example, can be 3.0 mV, 3.5 mV, 4.0 mV, 4.5 mV, 5.0 mV, 5.3 mV, 5.5 mV, 5.7 mV, 5.9 mV, 6.0 mV, 6.1 mV, 6.3 mV, 6.5 mV, 7.0 mV, 7.5 mV, 8.0 mV, preferably 5.5-6.5 mV, more preferably 6.1 mV.

[0022] In some embodiments, the preparation method of the composition is as follows:

[0023] An expression cassette expressing the CXCL3 protein was inserted into the pUC57-Kan plasmid, which comprises, from 5' end to 3' end, in order: a promoter site of T7 RNA polymerase, 5' UTR, "Kozak sequence + DNA sequence of CXCL3 shown in SEQ ID NO: 1", 3' UTR, poly A tail, and cleavage site of BspQI restriction enzyme.

[0024] After amplification and linearization using BspQI restriction enzyme, the in vitro transcription template expressing CXCL3 was recovered. The in vitro transcription was performed with the addition of ARCA (NEB, S1411S) for capping, obtaining the CXCL3 mRNA.

[0025] Encapsulation of CXCL3 mRNA: The liposome comprises ionizable lipid, non-cationic lipid, cholesterol, and PEG-modified lipid. The ionizable lipid ALC-0315, the non-cationic lipid distearoylphosphatidylcholine, cholesterol, and the PEG lipid ALC-0159 were mixed in ethanol, and the CXCL3 mRNA was encapsulated by a microfluidic device to obtain the lipid nanoparticle of CXCL3 mRNA.

[0026] In a second aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the composition of the first aspect and one or more pharmaceutically acceptable carriers.

[0027] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a small molecule drug, a nucleic acid drug, an antibody, an antibody fragment, a peptide, and / or an aptamer for treating a liver disease.

[0028] In a third aspect, the present disclosure provides use of the aforementioned mRNA composition or the aforementioned pharmaceutical composition in the manufacture of a medicament or a kit for chemotaxis of macrophages and / or treating a liver disease.

[0029] In a fourth aspect, the present disclosure provides use of an mRNA encoding CXCL3 in the manufacture of a medicament or a kit for chemotaxis of macrophages and / or treating a liver disease.

[0030] In some embodiments, the medicament or the kit comprises a therapeutically effective amount of an mRNA comprising a coding sequence of CXCL3, and a lipid nanoparticle, wherein the mRNA is comprised in the lipid nanoparticle.

[0031] In some embodiments, the lipid nanoparticle comprises an ionizable lipid, a non-cationic lipid, cholesterol, and a PEG lipid.

[0032] In some embodiments, the ionizable lipid is selected from (4-hydroxybutyl) aminobis(hexane-6,1 -diiyl) bis(2-hexyldecanoate) (ALC-0315, CAS No.: 2036272-55-4), 1,1 '-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1 -yl)ethyl)amino)bis(dodecan-2-ol) (C12-200), 4-(N,N-dimethylamino)butanoic acid-6,9,28,31 -tetraen-19-triacontanol ester (MC3), 1,2-dioleoyl-N,N-dimethyl-3-aminopropane (DLinDMA), 2,2-dioleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2DMA), (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1 H-cyclopenta[a]phenanthren-3-yl 3-(1 H-imidazol-4-yl)propanoate ethyl ester (ICE), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1 -yl) tetracos-15,18-dien-1 -amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1 -yl) tetracos-4,15,18-triethylenetetraamine-1 -amine (HGT5001 ), 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1 -yl]amino]butyl]-2,5-piperazinedione (OF-02), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1 -(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 5-carboxyspermyl glycine-dioctadecanamide (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1 -(cis,cis-9,12-octadecenyl)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β- yloxy)-3'-oxapentyl]-3-dimethyl-1-(cis,cis-9',1-2'-octadecenyl)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'- dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyl- oxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamoyl-3- dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamoyl (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminooxylethyl-[1,3]-dioxolane (DLin-K- XTC2-DMA), 2-((2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)- N,N-dimethylethanamine (HGT4003). In some preferred embodiments, the ionizable lipid is ALC-0315.

[0033] In some embodiments, the non-cationic lipid is selected from one or more of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)- cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2- oleoylphosphatidyethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (trans DOPE). In some preferred embodiments, the non-cationic lipid is distearoylphosphatidylcholine.

[0034] In some embodiments, the PEG lipid is selected from one or more of PEG-phospholipids and PEG-ceramides, for example from one or more of methoxypolyethylene glycol bimyristyl acetamide (ALC-0159), dimyristylglycerol-PEG2000 (DMG-PEG2000), distearoylphosphatidylethanolamine-PEG2000 (DSPE-PEG2000), dipalmitoylphosphatidylethanolamine-PEG2000 (DPPE-PEG2000), distearoylphosphatidylethanolamine-PEG2000 (DMPE-PEG2000), dioleoylphosphatidylethanolamine-PEG2000 (DOPE-PEG2000), distearoylphosphatidylethanolamine-PEG1000 (DSPE-PEG1000), dipalmitoylphosphatidylethanolamine-PEG1000 (DPPE-PEG1000), dimyristoylphosphatidylethanolamine-PEG1000 (DMPE-PEG1000), dioleoylphosphatidylethanolamine-PEG1000 (DOPE-PEG1000), distearoylphosphatidylethanolamine-PEG550 (DSPE-PEG550), dipalmitoylphosphatidylethanolamine-PEG550 (DPPE-PEG550), dimyristoylphosphatidylethanolamine-PEG550 (DMPE-PEG550) PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, PEG550-ceramide. In some embodiments, the PEG lipid is ALC-0159.

[0035] In some embodiments, the lipid nanoparticle comprises the ionizable lipid ALC-0315, distearoylphosphatidylcholine, cholesterol, and the PEG lipid ALC-0159.

[0036] In some embodiments, the lipid nanoparticle has a particle size of 85-120 nm, for example can be 85 nm, 86 nm, 87 nm, 88 nm, 90 nm, 92 nm, 94 nm, 96 nm, 98 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, preferably 88-89 nm, more preferably 88 nm.

[0037] In some embodiments, the monodispersity of the lipid nanoparticle is 0.030-0.099, for example, can be 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.063, 0.065, 0.067, 0.070, 0.073, 0.075, 0.077, 0.080, 0.083, 0.085, 0.087, 0.090, 0.095, 0.099, preferably 0.065-0.085, more preferably 0.075.

[0038] In some embodiments, the zeta potential of the lipid nanoparticle is 3.0-8.0 mV, for example, can be 3.0 mV, 3.5 mV, 4.0 mV, 4.5 mV, 5.0 mV, 5.3 mV, 5.5 mV, 5.7 mV, 5.9 mV, 6.0 mV, 6.1 mV, 6.3 mV, 6.5 mV, 7.0 mV, 7.5 mV, 8.0 mV, preferably 5.5-6.5 mV, preferably 5.5-6.5 mV, more preferably 6.1 mV.

[0039] In some embodiments, the amino acid sequence of the CXCL3 is as set forth in SEQ ID NO: 2.

[0040] In some embodiments, the mRNA comprises a CXCL3 coding region comprising the nucleotide sequence set forth in SEQ ID NO: 5 or comprising a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 5; in some preferred embodiments, the nucleotide sequence of the CXCL3 coding region is as set forth in SEQ ID NO: 5.

[0041] In some embodiments, the mRNA further comprises a 5' cap structure, a 5' UTR, a Kozak sequence, a 3' UTR, and / or a poly-A region.

[0042] In some embodiments, the 5' UTR sequence is the sequence set forth in SEQ ID NO: 6.

[0043] In some embodiments, the 3' UTR sequence is the sequence set forth in SEQ ID NO: 8.

[0044] In some embodiments, the mRNA comprises the nucleotide sequence set forth in SEQ ID NO: 4 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 4; in some preferred embodiments, the nucleotide sequence of the mRNA is set forth in SEQ ID NO: 4.

[0045] In some embodiments, the pharmaceutical or kit further comprises one or more pharmaceutically acceptable carriers.

[0046] In some embodiments, the pharmaceutical or kit further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a small molecule drug, a nucleic acid drug, an antibody, an antibody fragment, a peptide, and / or an aptamer for treating a liver disease.

[0047] In some embodiments, the mRNA is administered at an amount of 0.1-1.5 mg / kg per time, for example, 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.5 mg / kg, preferably 0.3-1.2 mg / kg per time, more preferably 1.0 mg / kg per time.

[0048] In some embodiments, the mRNA composition is administered 1-5 times per treatment cycle, for example, 1 time, 2 times, 3 times, 4 times, 5 times, preferably 3-4 times, more preferably 3 times.

[0049] In some embodiments, the liver disease comprises a liver fibrosis related disease or symptom; the liver fibrosis comprises cirrhosis caused by progression of liver fibrosis.

[0050] Hepatic (or liver) fibrosis is a complex, progressive process of diffuse, excessive deposition of scar tissue within the liver, resulting from ongoing inflammation and hepatocyte death that occurs in most types of chronic liver disease, which can cause structural destruction and alteration of the liver and can further evolve into cirrhosis and even liver failure. Many patients with liver disease, including chronic hepatitis, alcoholic hepatitis, non-alcoholic fatty liver disease, etc., after effective treatment, the liver is repaired by fibrosis, slowly progresses to liver fibrosis and cirrhosis. Non-alcoholic fatty liver disease is an umbrella term that encompasses a continuum of liver conditions that differ in the severity of the injury and the resulting fibrosis. Among them, isolated hepatic steatosis (fatty liver) is generally referred to as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis is usually defined as a more severe process with inflammation and hepatocyte damage (steatohepatitis), which is usually accompanied by fibrosis and often progresses to cirrhosis. Alcoholic liver disease refers to progressive liver disease caused by excessive long-term drinking, including alcoholic fatty liver (alcoholic steatosis), alcoholic hepatitis, liver fibrosis and cirrhosis.

[0051] In some embodiments, the liver fibrosis-related disease or condition includes chronic hepatitis B, chronic hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease, and diseases or conditions caused by liver fibrosis. The chronic hepatitis B, chronic hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease progresses or is accompanied by liver fibrosis or cirrhosis. The diseases or conditions caused by liver fibrosis include liver fibrosis-induced hepatorenal syndrome, liver fibrosis-induced liver function metabolic disorder, liver fibrosis-induced primary peritonitis, liver fibrosis-induced autoimmune hepatitis, liver fibrosis-induced primary sclerosing cholangitis, liver fibrosis-induced ascites, liver fibrosis-induced upper gastrointestinal variceal bleeding, and liver fibrosis-induced hepatic encephalopathy; liver fibrosis-induced: fatigue, weakness; loss of appetite, nausea, vomiting; chronic dyspeptic symptoms such as abdominal distension, constipation or diarrhea, dull pain in the liver area; chronic gastritis, such as acid reflux, belching, hiccups, upper abdominal pain and upper abdominal fullness, etc. Gastric symptoms; bleeding, chronic hepatitis, such as spider nevi, epistaxis, gingival bleeding, purpura or petechiae on the skin and mucosa, etc.; liver fibrosis-related infections, such as chronic hepatitis B, C and D, schistosomiasis, etc.; congenital metabolic defects, such as hepatolenticular degeneration, hemochromatosis, alpha 1-antitrypsin deficiency, etc.; chemical toxicity (such as chronic alcoholic liver disease and chronic drug-induced liver disease) and autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis; liver fibrosis-related: activation of hepatic stellate cells, extracellular matrix accumulation mediated by activation of hepatic stellate cells, increased serum aminotransferase levels, liver tissue inflammatory response, etc.

[0052] Fifthly, this disclosure provides mRNA encoding CXCL3 and compositions thereof for chemotaxis of macrophages and / or treatment of liver fibrosis and / or cirrhosis. The mRNA and compositions thereof are as described above.

[0053] Sixthly, this disclosure provides a method for chemotactic macrophages and / or treating liver fibrosis and / or cirrhosis, comprising administering an effective amount of mRNA encoding CXCL3 to a subject. In some embodiments, the mRNA is located within lipid nanoparticles, and the mRNA and the lipid nanoparticles are as described above.

[0054] In some embodiments, the administration comprises delivering an effective amount of the disclosed composition, pharmaceutical composition, or drug to a subject via any route for the purpose of its intended function, and may be administered via any suitable route, including but not limited to intravenous injection, subcutaneous injection, or intramuscular injection. In some embodiments, the disclosed mRNA, composition, pharmaceutical composition, or drug containing the CXCL3 coding sequence is administered to a subject in a single or multiple doses. In some embodiments, the disclosed mRNA, composition, pharmaceutical composition, or drug containing the CXCL3 coding sequence is administered once, twice, three times, four times, five times, six times, or seven times per week, for example, for one month, two months, three months, or four months; or once every two weeks, three weeks, or four weeks (e.g., for six months, ten months, or twelve months); or treatment is discontinued after one cycle of continuous administration and restarted after several months; or it may be used as chronic treatment, i.e., indefinite administration.

[0055] One or more technical solutions in the embodiments of this disclosure have at least the following technical effects or advantages:

[0056] The mRNA-lipid nanoparticle complex prepared in this disclosure significantly reduced the level of liver fibrosis, improved pseudolobules, and decreased liver damage in mice with liver cirrhosis after treatment. Serum ALT and AST levels also decreased. This indicates that this mRNA-lipid nanoparticle complex has potential application value in the in vivo delivery of nucleic acid therapeutic agents. Attached Figure Description

[0057] Figure 1 The results of denaturing agarose gel electrophoresis of the prepared CXCL3 mRNA sample are shown.

[0058] Figure 2 The expression and distribution of luciferase in healthy (WT) and liver fibrosis (TAA) mice injected with luciferase mRNA lipid nanoparticles are shown.

[0059] Figure 3The results of immunofluorescence detection of GFP expression location and main cell markers in the liver of healthy mice (WT) and liver fibrosis mice (TAA) injected with GFP mRNA lipid nanoparticles are shown.

[0060] Figure 4 The aggregation of F4 / 80-positive macrophages in the liver of fibrosis mice on the second day after the last injection of lipid nanoparticles (LNP) or CXCL3 mRNA lipid nanoparticle complexes (LNP-CXCL3) is shown.

[0061] Figure 5 The detection results of mouse liver 14 days after the last injection of lipid nanoparticles (LNP) or CXCL3 mRNA lipid nanoparticle complexes (LNP-CXCL3) are shown, wherein, Figure 5 A is the gross view of liver tissue; Figure 5 B is the result of Sirius red staining of the liver; Figure 5 C is the result of liver tissue hydroxyproline analysis.

[0062] Figure 6 The detection results of mouse serum and each main organ (liver, kidney, spleen, lung) 14 days after CXCL3 mRNA lipid nanoparticle complex treatment are shown, wherein, Figure 6 A is the gross view of liver tissue; Figure 6 B is the gross view of the spleen;

[0063] Figure 6 C is the serum biochemical (ALT, AST, CREA, UREA) level of the mouse; Figure 6 D is the HE staining result of each main organ. DETAILED DESCRIPTION

[0064] To make the objectives, technical solutions, and advantages of the embodiments of the present disclosure clearer, the technical solutions of the embodiments of the present disclosure will be described clearly and completely below with reference to the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are some but not all of the embodiments of the present disclosure. Based on the described embodiments of the present disclosure, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present disclosure.

[0065] The present disclosure can be implemented in other specific forms without departing from the essential attributes of the present disclosure. It should be understood that any and all embodiments of the present disclosure can be combined with the technical features in any other embodiment or multiple other embodiments to obtain additional embodiments without conflict, so long as the combinations do not depart from the essential attributes of the present disclosure. The present disclosure includes such additionally obtained embodiments.

[0066] All publications and patents mentioned in the present disclosure are hereby incorporated by reference in their entirety into the present disclosure. To the extent that any publication or patent incorporated by reference contradicts any disclosure herein, the disclosure herein will control.

[0067] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0068] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the disclosure. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will include the plural and vice versa.

[0069] Unless the context clearly indicates otherwise, as used herein the singular forms "a," "an," and "the" include the plural forms "at least one," "at least one," "one or more," and "two or more." For example, reference to "a cell" includes a plurality of such cells, as well as equivalents thereof known to those skilled in the art, and so forth.

[0070] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0071] Numerical ranges used herein should be read as if "to" was expressly written after each term, such as "1 to 20" should be read as meaning "from 1 to 20" or "from 1 and including 20" or "from the lower limit to the upper limit, i.e. 1 to 20." The use of "at least" with respect to a numerical range should be read as meaning "from the lower limit to infinity," unless explicitly stated otherwise.

[0072] As used herein, the terms "comprises," "comprising," "includes," "including" and the like can be used to signify the inclusion of one or more steps or integers or groups of steps or integers having attinention to any group preceeding those steps, integers, groups thereof or any other steps, integers, groups thereof, but not to the exclusion of any other steps, integers, groups thereof or steps, integers, groups thereof which can occur hefore any steps, integers, groups thereof. As used herein, the term "consisting of has the meaning ascribed in USN Pat. Code, and the term "consisting of means an closed collection of steps, integers, groups thereof or other features where the collection can include only the capture steps, integers, groups or other features. As used herein, the term "consisting essentially of has the meaning ascribed in USN Pat. Code and the term "consisting essentially of means a collection of steps, integers, groups thereof or other features in which in addition the

[0073] As used herein, the term "optional," "any," "any of," or "any one of" means that the event or circumstance subsequently described can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not. "One" and "an" are used in the present disclosure to refer to one or more than one of the grammatical object of the article.

[0074] As used herein, "and / or" is to be taken as specific disclosure of each of the two specified options, or of each combination of the two specified options.

[0075] As used herein, the term "nucleic acid" includes RNA, DNA, and cDNA molecules. It will be appreciated that, as a result of the degenerative nature of the genetic code, a large number of nucleotide sequences can give rise to a given protein. "Nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence" are used interchangeably and refer to a contiguous sequence of nucleic acids. The sequence can be single-stranded or double-stranded DNA or RNA, e.g., mRNA. The term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. The mRNA can contain one or more coding and non-coding regions. The mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, transcribed in vitro, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, the mRNA can comprise nucleotide analogs, e.g., analogs having chemical modifications of the bases or sugars, backbone modifications, etc. Unless otherwise indicated, mRNA sequences are presented in the 5' to 3' direction.

[0076] As used in the present disclosure, "5' cap structure" refers to a special modified structure attached to the 5' end of an mRNA molecule. It is usually composed of a 7-methylguanylate and is linked to the 5' end of the mRNA through a triphosphate chain.

[0077] As used in the present disclosure, "poly(A) tail" is typically a long sequence of cytosine nucleotides, typically about 25 to about 400 adenosine nucleotides, preferably about 50 to about 200 adenosine nucleotides, more preferably about 100 to about 150 adenosine nucleotides, or even more preferably about 120 to about 130 adenosine nucleotides. In one particular embodiment, the poly(A) tail described in the present disclosure contains 125 adenosine nucleotides.

[0078] As used in the present disclosure, 3' untranslated region (3'UTR) is typically a part of an mRNA that is located between the protein-coding region (i.e., open reading frame) and the poly(A) sequence of an mRNA. The 3'UTR of an mRNA is not translated into an amino acid sequence. The 3'UTR sequence is usually encoded by the gene that is transcribed into the corresponding mRNA during the process of gene expression.

[0079] The 5' untranslated region (5' UTR) referred to in the present disclosure is typically understood as a specific section of a messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Typically, the 5' UTR starts at the transcription start site and ends one nucleotide before the start codon of the open reading frame. The 5' UTR can comprise elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be, for example, a ribosomal binding site or a 5' end oligopyrimidine tract. The 5' UTR can be post- transcriptionally modified, for example, by the addition of a 5' cap.

[0080] In some embodiments, the mRNA further contains one or more sequences with regulatory functions. For example, in some specific embodiments, the mRNA further contains a functional sequence that facilitates the initiation of translation, such as a Kozak sequence, a Shine-Dalgarno sequence, a TISU (Translation Initiation Stimulation Element) sequence, or an optimized 5' UTR sequence (such as UTR7), etc. For another example, in some specific embodiments, the mRNA further contains a sequence that regulates mRNA stability and translation efficiency, such as a Chi-beta globin sequence, an AU-rich element (ARE) sequence, an internal ribosome entry site (IRES) sequence, a MicroRNA (miRNA) Binding Site, etc. In practice, one of skill in the art can identify the specific sequence and the placement of the above-mentioned sequences in combination with common sense.

[0081] As used herein, the terms "peptide," "polypeptide," "protein," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coding and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The point of origin of a protein or polypeptide is referred to as the "N-terminus" (or amino terminus, NH2 terminus, N-terminal, or amine terminus), which refers to the free amine (-NH2) group of the first amino acid residue of the protein or polypeptide. The terminus of a protein or polypeptide is referred to as the "C-terminus" (or carboxyl terminus, carboxy terminus, C-terminal, or COOH terminus), which refers to the free carboxyl (-COOH) of the last amino acid residue of the protein or peptide.

[0082] As used herein, “nucleic acid construct” refers to an artificially constructed nucleic acid fragment, particularly a DNA fragment, which is intended to be “transplanted” into a target cell (e.g., a bacterial cell) to modify the expression of a genomic gene or to express a gene / DNA sequence that can be included in the construct. DNA constructs can be linear or circular, and linear or circular DNA constructs that are integrated into the host bacterial genome or expressed in a plasmid are referred to interchangeably herein as “expression cassettes.”

[0083] As used herein, the terms “coding region” and “region encoding” and grammatical variations thereof refer to an open reading frame (ORF) in a polynucleotide that, upon expression, produces a polypeptide or protein.

[0084] As used herein, “expression” of a nucleic acid sequence refers to translation of mRNA into a polypeptide, assembly of multiple polypeptides into an intact protein (e.g., enzyme), and / or post-translational modification of a polypeptide or fully assembled protein (e.g., enzyme). In this document, the terms “expression” and “production” and grammatical equivalents are used interchangeably.

[0085] While the present disclosure sets forth some specific amino acid sequences or nucleotide sequences, e.g., as set forth in the sequence listing, it is understood that one specific amino acid sequence or nucleotide sequence described in the present disclosure includes conservatively modified variants thereof, e.g., sequences that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% homologous thereto, so long as the biological function or activity of the specific amino acid sequence or nucleotide sequence is not lost.

[0086] As used herein, the term "homology" has its art-recognized meaning and is a central concept in comparative biology. At its most basic, homology refers to the fact that two samples being compared (e.g., a stretch of amino acid sequence or a stretch of nucleotide sequence) share a common ancestor. Generally, two traits (states) in two species can be said to be homologous if either of two conditions is met: 1) they are identical to a trait found in an ancestral group of those species; and 2) they are different traits that have an ancestor-descendant relationship. Nucleotide or amino acid sequence homology (%) can be determined initially using programs commonly used in the art (e.g., BLAST). As used herein, the term "homology" refers to the exact nucleotide-nucleotide or amino acid-amino acid correspondence, respectively, between two polynucleotides or amino acid sequences. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "homology (%)." Whether nucleic acid or amino acid sequences, the homology (%) of two sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100.

[0087] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refers to those nucleic acids that encode identical or conservatively modified variants of the amino acid sequences due to the degeneracy of the genetic code. Numerous functionally identical nucleic acids encode any given protein due to the degeneracy of the genetic code. For instance, the codons five (and therefore, the corresponding DNA codons, T is substituted for U) are interchangeable to code for each specific amino acid, e.g., the codons GCA, GCC, GCG and GCU all encode alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons and still specify alanine. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Those of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and UGG which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each of the silent variations of a nucleic acid which encodes a polypeptide of the present disclosure is a conservatively modified variant of the disclosed polypeptide sequences, and such variations are included in the present disclosure.

[0088] For amino acid sequences, the skilled artisan will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or small number of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, for instance, a change at a position where the amino acid residue is selected from any one of the integers between 1 and 15 can be made. For example, 1, 2, 3, 4, 5, 7 or 10 changes can be made. Conservatively modified variants typically provide biological activity similar to the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzymatic activity, or ligand / receptor binding capacity is typically at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein with its natural substrate. The skilled artisan is well aware of amino acid conservations that provide functionally similar substitutions. For example, modifications can be introduced into the sequences set forth herein by standard techniques known in the art, e.g., gene synthesis and PCR-mediated mutagenesis. Conservative modifications include "conservative substitutions," which are substitutions of amino acids with similar properties, e.g., replacement of one amino acid with another having similar side chain properties, such that the secondary structure and hydrophilicity properties of the polypeptide are not substantially altered. Families of amino acid residues having similar side chains are well known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0089] As used herein, the terms "lipid nanoparticle," "LNP," and "LNPs" are applied interchangeably and can comprise any lipid capable of forming a particle to which one or more nucleic acid molecules are attached or in which one or more nucleic acid molecules are encapsulated. Herein, a lipid nanoparticle in which one or more nucleic acid molecules are encapsulated can be referred to as a nucleic acid lipid nanoparticle composition, and can also be referred to as a nucleic acid lipid nanoparticle complex. The term "lipid" refers to a group of organic compounds that are derivatives (e.g., esters) of fatty acids and are typically characterized by being insoluble in water but soluble in many organic solvents. The lipid nanoparticle refers to any particle or small vesicle having a nanoscale size formed from one or more lipid components.

[0090] As used herein, the terms “ionizable lipid,” “cationic lipid,” and “ionizable cationic lipid” are used interchangeably to refer to a lipid molecule that is capable of carrying a positive charge under physiological pH conditions. Cationic liposomes readily associate with negatively charged nucleic acids, i.e., by electrostatic forces with the negatively charged phosphate groups present in the nucleic acid.

[0091] As used herein, the terms “non-cationic lipid” and “neutral lipid” are used interchangeably to refer to a helper lipid that is not charged or exists as a zwitterion at the selected pH. Non-cationic lipids can modulate nanoparticle fluidity into a lipid bilayer structure and improve efficiency by facilitating lipid phase transition, while also potentially influencing target organ specificity.

[0092] As used herein, the term “polyethylene glycol lipid” (PEG lipid) refers to a lipid molecule comprising a lipid moiety and a polyethylene glycol moiety.

[0093] Various methods of lipid nanoparticle preparation are suitable for the present disclosure. For example, ethanol dilution, freeze-thaw, thin film hydration, sonication, extrusion, high pressure homogenization, detergent dialysis, microfluidization, tangential flow diafiltration, sterile filtration, and / or lyophilization can be used.

[0094] As used herein, the term “particle size” or “particle diameter” refers to the average diameter of particles in a sample as measured by dynamic light scattering (DLS), multi-angle light scattering (MALS), nanoparticle tracking analysis, or similar techniques. It will be appreciated that the dispersion of the lipid nanoparticles described herein is not uniform in size, but can be described by an average diameter and optionally a polydispersity index.

[0095] As used herein, the term “encapsulation efficiency” represents the proportion of nucleic acid that is encapsulated inside the lipid nanoparticle out of the total nucleic acid. Encapsulation efficiency is typically detected using fluorescence spectrophotometry. As an example with RNA, first the amount of RNA that is free outside the lipid nanoparticle is detected using RiboGreen fluorescent dye in the LNP-RNA solution. Then the lipid nanoparticle structure is disrupted using Triton-100, which releases the RNA to the outside solution. The total amount of RNA in the solution is then detected. The difference between the two is the amount of RNA that is encapsulated inside the LNP particle, and thus the encapsulation efficiency is obtained.

[0096] As described herein, the term "zeta potential" (zeta potential) refers to the overall surface charge acquired by a nanoparticle in a particular medium (e.g., water) and is a measure of electrostatic attraction and repulsion. Zeta potential values are indicative of dispersion stability, aggregation, and diffusion behavior. Zeta potential can be calculated from electrokinetic data obtained from, e.g., laser Doppler velocimetry. In this technique, a voltage is applied across a cell containing a dispersion of nanoparticles between a pair of electrodes. The charged nanoparticles are attracted to the oppositely charged electrode, their velocity is measured and expressed in units of field strength as their electrophoretic mobility. Zeta values can predict the permeation of various cell membranes. The zeta potential of a lipid nanoparticle can be measured by a Zetasizer Pro (an instrument from Malvern Instruments Ltd). The LNP can be diluted in a pH buffer (e.g., PBS pH = 7.4) to a certain level (e.g., 1.0 ng / pL) of total mRNA and then loaded into a disposable folded capillary cell (e.g., DTS1070). The sample can be equilibrated for a period of time (e.g., 120 seconds) and the measurement is repeated multiple times with a period of time (e.g., 20 seconds) between measurements.

[0097] As used herein, the term "monodispersity" refers to the uniformity of distribution of particles or granules in size, shape, and structure. If the particle size distribution of a nanoparticle system is very narrow, i.e., the size of most particles is almost equal, then the system is considered monodisperse. A high monodispersity index means better consistency in particle size.

[0098] As used herein, the term "encapsulate" refers to enclosing, surrounding, or enveloping.

[0099] As used herein, the term "deliver" refers to providing an entity to a target. For example, delivering a polynucleotide to a subject can involve administering a nanoparticle composition comprising the polynucleotide to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or a mammalian cell can involve contacting one or more cells with the nanoparticle composition.

[0100] As used herein, the term "chemotaxis" refers to the positive (toward the stimulus) or negative (away from the stimulus) migration of a cell induced by a chemical stimulus, and in particular to the positive migration. The chemotactic macrophage refers to the migration of a macrophage induced by a chemical stimulus.

[0101] As used herein, the term "aptamer" refers to a polynucleotide, typically RNA or DNA, that has useful biological activity in terms of biochemical activity, molecular recognition, or binding properties. Typically, aptamers have molecular activities such as binding to a target molecule at a specific epitope (region).

[0102] As used herein, the term "treatment" is intended to include prophylaxis and therapy, methods in which a beneficial or desired result is achieved, including clinical results, for the purposes of the present application, a beneficial or desired result includes, but is not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, diminishment of extent of disease, stabilization of disease (e.g., preventing or delaying exacerbation of disease), preventing or delaying spread (e.g., metastasis) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, providing a remission from (partial or total) disease, decreasing the dose of one or more other medications required to treat the disease, delay of disease progression, increasing or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also includes reduction in pathological outcomes (e.g., tumor volume) of cancer. In the context of cancer, "treatment" includes any or all of: inhibiting growth of cancer cells, inhibiting replication of cancer cells, reducing overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0103] As used herein, the term "effective amount" or "therapeutically effective amount" includes an amount that is sufficient to ameliorate or prevent a symptom or condition of a medical disorder. An effective amount also means an amount that is sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the patient, the method route and dose of administration, and the severity of side effects. The effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0104] As used herein, the terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, apes, humans, farm animals, sport animals, and pets.

[0105] The amount of drug administered can depend on the subject being treated, the age, health, sex, and weight of the subject, kind of concurrent treatment, if any, the severity of the condition, the nature of the effect desired, the mode and frequency of administration, and the judgment of the prescribing physician. The frequency of administration can also depend on the pharmacodynamic effects on arterial partial pressure of oxygen. However, the optimal dose can be adjusted according to individual subject, as is understood by those skilled in the art and without undue experimentation. This will generally include adjustment of standard dosages (e.g., if the patient is small, the dose is reduced).

[0106] As used herein, the term "pharmaceutical composition" refers to a mixture of one or more components. The purpose of a pharmaceutical composition is to facilitate administration of a compound of the disclosure to an organism. Pharmaceutical compositions of the disclosure can include one or more pharmaceutically acceptable salts, pharmaceutically acceptable carriers and / or adjuvants, such as antioxidants, preservatives, wetting agents, emulsifying agents and dispersing agents, etc.

[0107] As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation, formulation auxiliary or carrier with which the therapeutic agent is administered, which together with the active agent, makes up a "pharmaceutical composition" for administration to an individual. The pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is suitable for the formulation employed. Suitable carriers are well known to those skilled in the art, such as carbohydrates, waxes, water soluble and / or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, liposomes, polymeric micelles, or inorganic nanocarriers, and the like.

[0108] The "pharmaceutical composition" of the present disclosure can also be administered to a patient or subject in need of such treatment in any suitable administration manner, such as oral, intravenous, parenteral, rectal, pulmonary, or topical administration, etc. When used for oral administration, the pharmaceutical composition can be formulated into oral preparations, such as oral solid preparations, such as tablets, capsules, pills, granules, etc.; or, oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When formulated into oral preparations, the pharmaceutical preparation can further comprise suitable fillers, binders, disintegrants, lubricants, etc.

[0109] The pharmaceutical composition of the present disclosure can be formulated into any pharmaceutically acceptable dosage form for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration, for example, can be formulated into tablets, lozenges, capsules, pills, solutions, suspensions, syrups, injections, suppositories, inhalants, or sprays.

[0110] As used herein, the term "kit" refers to a package of one or more components, which can be individually packaged or placed in a container, such as a tube, bottle, vial, pouch, blister, syringe, or other suitable container device. The kits described herein can also include instructions for carrying out the subject methods using the components of the kit (e.g., instructions for preparing and / or using the pharmaceutical compositions described herein). The instructions for carrying out the subject methods are typically recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. As such, the instructions can be present in the kit package in the form of printed matter, in the container of the kit or its components (i.e., components associated with the packaging or sub-packaging). In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium (e.g., a CD-ROM, disk, etc.). In other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via an internet site, are provided.

[0111] As used herein, the terms "fibrosis" and "fibrotic condition" are used interchangeably herein and refer to pathological tissue healing in which connective tissue replaces normal parenchymal tissue, leading to substantial tissue remodeling and permanent scar tissue formation. Fibrosis can occur in any of multiple organs, including but not limited to the liver, lung, kidney, skin, eye, or myocardium. As used herein, the term fibrosis or fibrotic condition also refers to different stages of tissue fibrosis.

[0112] As used herein, the term "liver disease" refers to a pathological condition that occurs in the liver. The term "cirrhosis of the liver" is also known as liver cirrhosis and refers to the formation of fibrous tissue (fibrosis) in the liver in place of liver cells that have died. Cirrhosis is an end-stage liver disease characterized by damage due to liver disease that leads to the formation of fibrotic scar tissue and causes impaired liver function. Liver damage causes tissue repair and subsequent generation of scar tissue, which over time replaces normal functioning tissue, leading to decreased liver function and eventually development of cirrhosis.

[0113] As used herein, the term "therapeutic agent" refers to an 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.

[0114] As used herein, the term "CXCL3" refers to any native CXCL3 (Chemokine (C-X-C motif) ligand 3; also known as Macrophage Inflammatory Protein 2-beta (MIP2-beta)) from any vertebrates source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CXCL3 as well as any form of CXCL3 that results from processing in the cell. The term also encompasses naturally occurring variants of CXCL3, e.g., splice variants or allelic variants.

[0115] For the purpose of the present disclosure, technical solutions and advantages, the following will be further described in detail in combination with embodiments. The specific conditions are not specified in the embodiments, and the conventional conditions or the conditions recommended by the manufacturer are used. All reagents or instruments are not specified by the manufacturer, and are all conventional products that can be purchased in the market. In order to better illustrate the present disclosure, numerous specific details are given in the following specific embodiments. The specific embodiments described herein are only used to explain the present disclosure, and do not constitute any limitation on the present disclosure. In addition, in the following description, the description of the known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure.

[0116] Embodiments

[0117] Example 1. Preparation of CXCL3 mRNA Lipid Nanoparticles (CXCL3 mRNA-LNP)

[0118] 1. CXCL3 mRNA synthesis:

[0119] ① The DNA sequence of mouse (mus musculus) CXCL3 was obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ).

[0120] The CXCL3 DNA sequence (SEQ ID NO: 1) is as follows: ATGGCCCCTCCCACCTGCCGGCTCCTCAGTGCTGCACTGGTCCTGCTGCTGCTGCTGGCCACCAACCACCAGGCTACAGGGGCTGTTGTGGCCAGTGAGCTGCGCTGTCAGTGCCTGAACACCCTACCAAGGGTTGATTTTGAGACCATCCAGAGCTTGACGGTGACGCCCCCAGGACCCCACTGCACCCAGACAGAAGTCATAGCCACTCTCAAGGATGGTCAAGAAGTTTGCCTCAACCCCCAAGGCCCCAGGCTTCAGATAATCATCAAGAAGATACTGAAGAGCGGCAAGTCCAGCTGA.

[0121] The CXCL3 amino acid sequence (SEQ ID NO: 2) is as follows: MAPPTCRLLSAALVLLLLLATNHQATGAVVASELRCQCLNTLPRVDFETIQSLTVTPPGPH CTQTEVIATLKDGQEVCLNPQGPRLQIIIKKILKSGKSS.

[0122] 2) Design an expression cassette of CXCL3 mRNA, which is in the order of 5' end to 3' end: 5' UTR, "Kozak sequence + DNA sequence of Cxcl3", 3' UTR, poly A tail and cleavage site of BspQI restriction endonuclease, and the nucleotide sequence is as follows:

[0123] CCAGATCTCACCACAGCCCTTCGCACCAGCTCCCGCGCTCCAGACTCCAGCCACACT

[0124] CCAGCCTAGCGCCAGCCACCATGGCCCCTCCCACCTGCCGGCTCCTCAGTGCTGCAC

[0125] TGGTCCTGCTGCTGCTGCTGGCCACCAACCACCAGGCTACAGGGGCTGTTGTGGCCA

[0126] GTGAGCTGCGCTGTCAGTGCCTGAACACCCTACCAAGGGTTGATTTTGAGACCATCC

[0127] AGAGCTTGACGGTGACGCCCCCAGGACCCCACTGCACCCAGACAGAAGTCATAGCC

[0128] ACTCTCAAGGATGGTCAAGAAGTTTGCCTCAACCCCCAAGGCCCCAGGCTTCAGATA

[0129] ATCATCAAGAAGATACTGAAGAGCGGCAAGTCCAGCTGAGCCGGGGAAAGGAGGAA

[0130] GCCCCTCACCATGATGAAGGACCTGCATTCTAAATCAGAGAAAAGCGATCCATCCCA

[0131] ACGGTGTCTGGATGTGTCTTTACCTGCTTGTTTTTAACAACTCCTGAGAGTTCATACCT

[0132] ATTTTATTTCACATGTAAAATAACTACGGGTATAATTGCATCTACTTGCAAGATGGCATC

[0133] TTATTTAATGTTTATTTAATATCAAGTCTAAGTTTAACCAAGCCTATTCTTAACTTGAAG

[0134] GTTAGAGATTTTCAGATTCTGTTAGGGAGTTATATTGCTATTTCTGAGGCAGTATTCCTT

[0135] GGCTGGCCATTTTGGTGGCAGCTGTGATAGTCAAAAAACAGACAAACAAGGTCATGC

[0136] CGGTGTAGGAAGAATGCATGTGCACATCTAGTTTTGTAACTATACAAGATTATCAGTTG

[0137] TTATTTATTGAAATGGTCTTACGGTGTCACATGAAATATCAACTTTGTGTTAAAGCTTT

[0138] AAGAATTATAATGCATTATATATTTCTTAGACATTTTAAGTGATTTTTTAAAAATAAGGC

[0139] ACAGTGACTATTTAATGCCTTTATGTCTTAGAATGGAGAAGTTTAAATATTTATTGGTAT

[0140] TTTTACAAATAATTTGAAAATAAAATATTTTAAAATAAAAAAAAAAAAAAAAAAAAA

[0141] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCTCTTC (SEQ ID NO: 3).

[0142] ③ Commissioned living organisms to synthesize the above sequence and clone to pUC57 vector, sequencing confirmation, successfully obtained in vitro transcription template, using T7 RNA synthesis kit (NEB, E2050S) in vitro transcription, in the process of adding ARCA (NEB, S1411S) capping, finally adding 5M LiCl solution mixed, placed in -20℃, incubated for 30 minutes, collect the precipitate. With 70% ethanol wash the precipitate, remove impurities, finally using enzyme-free water to dissolve the purified mRNA, get CXCL3 mRNA sample, after denaturing agarose gel electrophoresis detection, such as Figure 1 , the size of the mRNA obtained is consistent with the expected, successfully prepared CXCL3 mRNA.

[0143]

[0144] wherein the CXCL3 coding region mRNA sequence (SEQ ID NO: 5) is as follows: AUGGCCCCUCCCACCUGCCGGCUCCUCAGUGCUGCACUGGUCCUGCUGCUGCUGCUGGCCACCAACCACCAGGCUACAGGGGCUGUUGUGGCCAGUGAGCUGCGCUGUCAGUGCCUGAACACCCUACCAAGGGUUGAUUUUGAGACCAUCCAGAGCUUGACGGUGACGCCCCCAGGACCCCACUGCACCCAGACAGAAGUCAUAGCCACUCUCAAGGAUGGUCAAGAAGUUUGCCUCAACCCCCAAGGCCCCAGGCUUCAGAUAAUCAUCAAGAAGAUACUGAAGAGCGGCAAGUCCAGCUGA.

[0145] 5' cap is a CAP1 structure.

[0146] 5' UTR sequence (SEQ ID NO: 6) is as follows:

[0147] CCAGAUCUCACCACAGCCCUUCGCACCAGCUCCCGCGCUCCAGACUCCAGCCACACUCCAGCCUAGCGCCA.

[0148] Kozak sequence (SEQ ID NO: 7) is as follows:

[0149] GCCACC.

[0150] 3' UTR sequence (SEQ ID NO: 8) is as follows:

[0151] GCCGGGGAAAGGAGGAAGCCCCUCACCAUGAUGAAGGACCUGCAUUCUAAAUCAG

[0152] AGAAAAGCGAUCCAUCCCAACGGUGUCUGGAUGUGUCUUUACCUGCUUGUUUUU

[0153] AACAACUCCUGAGAGUUCAUACCUAUUUUAUUUCACAUGUAAAAUAACUACGGG

[0154] UAUAAUUGCAUCUACUUGCAAGAUGGCAUCUUAUUUAAUGUUUAUUUAAUAUCA

[0155] AGUCUAAGUUUAACCAAGCCUAUUCUUAACUUGAAGGUUAGAGAUUUUCAGAUU

[0156] CUGUUAGGGAGUUAUAUUGCUAUUUCUGAGGCAGUAUUCCUUGGCUGGCCAUUU

[0157] UGGUGGCAGCUGUGAUAGUCAAAAAACAGACAAACAAGGUCAUGCCGGUGUAGG

[0158] AAGAAUGCAUGUGCACAUCUAGUUUUGUAACUAUACAAGAUUAUCAGUUGUUAU

[0159] UUAUUGAAAUGGUCUUACGGUGUCACAUGAAAUAUCAACUUUGUGUUAAAGCUU

[0160] UAAGAAUUAUAAUGCAUUAUAUAUUUCUUAGACAUUUUAAGUGAUUUUUUAAAA

[0161] AUAAGGCACAGUGACUAUUUAAUGCCUUUAUGUCUUAGAAUGGAGAAGUUUAAAUAUUUAUUGGUAUUUUUACAAAUAAUUUGAAAAUAAAAUAUUUUAAAAUAA.

[0162] Poly A tail sequence (SEQ ID NO: 9) is as follows: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAA.

[0163] 2. Encapsulation:

[0164] The obtained CXCL3 mRNA above was dissolved in citrate buffer to a final concentration of 0.5 mg / mL, and the ionizable lipid ALC-0315, distearoylphosphatidylcholine, cholesterol, PEG lipid ALC-0159 were mixed in ethanol at a ratio of 50%:10%:38.5%:1.5% to obtain a lipid mixture with a final concentration of 10 mM, and then the mRNA was mixed with the lipid mixture by a nanoparticle preparation system (NanoAssembler Ignite, Cytiva) at a flow rate ratio of 3:1 and a total flow rate of 8 mL / min. After mixing, the obtained product was subjected to ultrafiltration to remove excess ethanol, and finally the lipid nanoparticles encapsulating CXCL3 mRNA (CXCL3 mRNA-LNP) were obtained.

[0165] The particle size, monodispersity and zeta potential of the lipid nanoparticles obtained above were measured by a laser particle size analyzer (Nano ZS, Malvern). At the same time, after the lipid nanoparticles were lysed with 0.2% Triton X-100, the mRNA encapsulation rate was determined by a Quant-iTRiboGreen RNA kit (R11490, Thermo Fisher), and the characterization results were as follows:

[0166] Particle size: 88.52 ± 0.459 nm;

[0167] Monodispersity: 0.0749 ± 0.010138;

[0168] Zeta potential: 6.101 ± 0.3981 mV;

[0169] mRNA encapsulation rate: 94.33%.

[0170] Example 2. Targeting detection of lipid nanoparticles (LNP)

[0171] 1. Targeting detection in fibrotic liver

[0172] C57 mice were fed with 250 mg / L TAA (thioacetamide) in drinking water for a total of 8 weeks to construct a liver fibrosis model. In this experiment, the mRNA contained in the LNP was Luciferase mRNA capable of expressing luciferase. The preparation method of Luciferase mRNA-LNP was the same as that of Example 1, except that the CXCL3 mRNA was replaced with Luciferase mRNA. Healthy mice were used as a control group, and liver fibrosis mice were injected with 40 μg of Luciferase mRNA-LNP via the tail vein, respectively. Eight hours after injection, the bioluminescence of the tissues of healthy mice and liver fibrosis mice was photographed by small animal live imaging technology. D-luciferin was injected intraperitoneally 10 minutes before the photographing, and the mice were anesthetized with isoflurane. The distribution of LNP-delivered mRNA in the two groups of mice was observed by Tian Neng small animal live imaging instrument.

[0173] When the Luciferase mRNA-LNP enters the mouse body and expresses luciferase, the luciferin substrate is given to the mouse. Under the catalysis of luciferase, the substrate emits light, and the small animal live imaging instrument detects the bioluminescence to reflect the expression amount and distribution of luciferase. This detection method can specifically judge the distribution and expression of mRNA.

[0174] The results are shown in Figure 2 , and the mRNA delivered by LNP is mainly expressed in the liver of mice. The liver fibrosis background does not affect the location of delivery and expression, and the expression efficiency.

[0175] 2. Cell location of the expression of the target protein in the fibrotic liver

[0176] In this experiment, the mRNA contained in the LNP was GFP mRNA capable of expressing green fluorescent protein (GFP), and the other experimental conditions and steps were the same as those in 2.1, except that the Luciferase mRNA was replaced with GFP mRNA. By immunofluorescence co-staining technology, the expression location of the main cell markers (Hnf4α, CK19, CD31, F4 / 80, α-sma) in the liver of mice and GFP was detected. The specific cells in which GFP mRNA was expressed in the liver of the control group and the fibrosis group of mice can be specifically judged.

[0177] The results are shown in Figure 3 , and the mRNA delivered by LNP is mainly expressed in the liver cells and kupffer cells of mice, and the liver fibrosis background does not affect the expression location or efficiency.

[0178] Example 3. CXCL3 mRNA-LNP for treating liver cirrhosis

[0179] 1. Construct cirrhosis model by TAA drinking water, the modeling method adopted is: 250 mg / L TAA is added to daily drinking water, and induction is performed for a total of 8 weeks. Tail vein injection is adopted for administration, and an equal volume of CXCL3 mRNA-LNP or blank LNP is used. The CXCL3 mRNA-LNP and blank LNP are obtained according to the preparation method of Example 1, except that the blank LNP does not encapsulate mRNA. The administration dose and administration time are as shown in Table 1. Healthy mice are used as a control group, normal drinking water is used, and no drug is administered.

[0180] Table 1. Administration scheme and detection of mice in each group in Example 3

[0181]

[0182] 2. After 48 hours after the last administration or 14 days after recovery, the serum and organ samples (liver, spleen, lung, kidney) of each group are collected, the liver tissue is subjected to HE staining and picrosirius staining, the macrophage marker F4 / 80 is detected by immunohistochemical staining; the liver tissue hydroxyproline is detected by a kit (elabscience, E-BC-K062-M), so as to evaluate whether macrophages are aggregated in the liver and whether the fibrosis level is relieved and recovered after administration.

[0183] The results are analyzed as follows:

[0184] As Figure 4 It is shown that a large number of F4 / 80 positive macrophages are aggregated in the liver of the mouse injected with the last dose of CXCL3 mRNA-LNP the next day.

[0185] As Figure 5 It is shown that the granular feeling on the surface of the liver of the treated mouse is reduced, and the picrosirius staining and hydroxyproline analysis show that the fibrosis level in the liver is significantly reduced.

[0186] Example 4. Safety of CXCL3 mRNA-LNP

[0187] Healthy mice are divided into two groups, each group has 3 mice, and 1 mg / kg of blank LNP (WT) or 1 mg / kg of CXCL3 mRNA-LNP (WT+LNP-CXCL3) is injected into the tail vein respectively. The preparation method of blank LNP and CXCL3 mRNA-LNP is the same as that of Example 3. Administration is performed 3 times, and the time interval is 24 hours each time. After the last injection, recovery is performed for 14 days, and the serum and organs of the mice are collected. The detection method is the same as that of Example 3, and in addition, a third-party detection agency (Lile Biological) is entrusted to detect the serum biochemistry (ALT, AST, CREA, UREA).

[0188] As Figure 6The results of HE staining of mouse liver, kidney, spleen, and lung showed that CXCL3 mRNA-LNP did not cause abnormal organs in mice. The results of serum ALT, AST, CREA, and UREA showed no damage to the liver and kidney function of mice.

Claims

1. The application of CXCL3 mRNA in the preparation of drugs for treating liver diseases, wherein, The mRNA is encapsulated in lipid nanoparticles; the liver disease is cirrhosis caused by liver fibrosis.

2. The use of an mRNA composition or a pharmaceutical composition comprising a therapeutically effective amount of said mRNA composition in the preparation of a medicament for treating liver diseases, wherein, The mRNA composition comprises mRNA and lipid nanoparticles, wherein the mRNA is encapsulated within the lipid nanoparticles, and the mRNA is capable of expressing CXCL3; the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers; the liver disease is cirrhosis resulting from liver fibrosis.

3. The application according to claim 1 or 2, wherein, The amino acid sequence of CXCL3 is shown in SEQ ID NO:

2.

4. The application according to claim 1 or 2, wherein the mRNA comprises a CXCL3 coding region, and the nucleotide sequence of the CXCL3 coding region is shown in SEQ ID NO:

5.

5. The application according to claim 1 or 2, wherein the mRNA further comprises a 5' cap structure, a 5' UTR, a Kozak sequence, a 3' UTR, and / or a poly-A tail.

6. The application according to claim 5, wherein the 5'UTR sequence is the sequence shown in SEQ ID NO:

6.

7. The application according to claim 5, wherein the 3'UTR sequence is the sequence shown in SEQ ID NO:

8.

8. The application according to claim 1 or 2, wherein the nucleotide sequence of the mRNA is as shown in SEQ ID NO:

4.

9. The application according to claim 1 or 2, wherein the lipid nanoparticles comprise ionizable lipid ALC-0315, distearate phosphatidylcholine, cholesterol, and PEG lipid ALC-0159.

10. In the application according to claim 1 or 2, the lipid nanoparticles have a particle size of 85-120 nm.

11. In the application according to claim 1 or 2, the lipid nanoparticles have a particle size of 88-89 nm.

12. In the application according to claim 1 or 2, the lipid nanoparticles have a particle size of 88 nm.

13. In the application according to claim 1 or 2, the dosage of the mRNA is 0.1-1.5 mg / kg per dose.

14. In the application according to claim 1 or 2, the dosage of the mRNA is 0.3-1.2 mg / kg per dose.

15. The application according to claim 1 or 2, wherein the amount of mRNA used is 1.0 mg / kg per dose.

16. The application according to claim 2, wherein the pharmaceutical composition further comprises an additional therapeutic agent.

17. The application according to claim 16, wherein the additional therapeutic agent comprises a small molecule drug, a nucleic acid drug, an antibody, and / or a peptide for treating cirrhosis resulting from the progression of liver fibrosis.

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