OTC mRNA nucleic acid medicine for treating hepatic fibrosis and hepatocellular carcinoma and application of OTC mRNA nucleic acid medicine

Abstract

The invention provides an mRNA nucleic acid medicine and application thereof. The mRNA comprises a nucleic acid with a nucleotide sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 3. The mRNA can be used for treating hepatic fibrosis, relieving clinical hepatic fibrosis, inhibiting the progress of hepatic fibrosis, promoting liver cancer cells to differentiate into hepatic cells with partial liver functions, recovering the liver functions and prolonging the survival time of HCC patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nucleic acid, in particular to an OTC mRNA nucleic acid drug for treating liver fibrosis and hepatocellular carcinoma and application thereof. BACKGROUND

[0002] Ornithine transcarbamylase (OTC) is expressed in the liver and intestinal mucosa, and is a mitochondria urea cycle rate-limiting enzyme. Some studies have shown that the accumulation of ammonia caused by the decrease of OTC activity can cause chronic liver damage, and is also a potential inducer of hepatocellular carcinoma (HCC). Ornithine transcarbamylase deficiency (OTCD) is the most common urea cycle disorder (UCD). As the second key enzyme in the urea cycle of mammalian liver, OTC transfers carbamoyl phosphate (CP) to the amino group of L-ornithine (ORN) in mitochondria to generate citrulline and phosphate. This anabolic two-substrate reaction is involved in many basic metabolic pathways, such as the biosynthesis of citrulline and arginine, ammonia balance and urea cycle. Therefore, the most common biochemical features of OTCD patients are severe hyperammonemia, decreased citrulline, elevated glutamine, and elevated aspartic acid and pyrimidine in urine. High blood ammonia and high glutamine in the brain have toxic effects on the central nervous system, which can cause changes in mental status, seizures, brain edema, and even death in severe cases. Acute hyperammonemia can also cause liver dysfunction. It has been reported that OTCD patients and mice can develop liver fibrosis / cirrhosis, and OTCD can also cause hepatocellular carcinoma. Therefore, restoring the expression of OTC in liver cells is an effective strategy for treating liver fibrosis and hepatocellular carcinoma.

[0003] The principle of clinical treatment for OTCD is to control diet, reduce protein intake, and cooperate with nitrogen scavenging drugs (such as sodium benzoate, sodium phenylbutyrate, etc.). One strategy is gene therapy delivered by adeno-associated virus (AAV), which can partially restore OTC enzyme activity in the liver of mice and reduce the excretion of orotic acid. When using AAV8-OTC for gene therapy of OTC-KO Het mice, not only can the underlying metabolic abnormalities be corrected, but also the development of liver fibrosis can be prevented. A phase I clinical trial (NCT02651675) evaluating the efficacy of AAV8-OTC is currently underway. The mRNA drug ARCT-810 for clinical trials of OTCD is undergoing phase I clinical trials (NCT04442347). AAV therapy can effectively correct ammonia metabolism for a long time and effectively prolong life, but a large number of liver nodules or tumors are observed in animals receiving treatment, indicating that AAV gene therapy also still has a risk of carcinogenesis. mRNA does not enter the nucleus, only expresses the target protein in the cytoplasm, and the mRNA itself does not have infectivity and can be degraded through normal cellular pathways, and has good safety. mRNA technology has been applied to vaccine preparation, gene editing therapy, cell therapy, and protein replacement therapy in many fields. Protein replacement therapy based on mRNA can solve the problem of protein deficiency, improve the low level of protein, or replace non-functional proteins with functional proteins.

[0004] However, there is no report on the treatment of liver fibrosis and hepatocellular carcinoma by OTC mRNA protein replacement strategy. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides an OTC mRNA nucleic acid drug and its application. It provides a new drug for treating liver fibrosis and / or hepatocellular carcinoma.

[0006] In one aspect of the present application, an mRNA is provided, which comprises at least one of the following 1)-10):

[0007] 1) a nucleic acid having a nucleotide sequence as shown in any one of SEQ ID NO: 1-3;

[0008] 2) a nucleic acid in which one or more nucleotides are substituted, deleted or added in the sequence of the nucleic acid of 1);

[0009] 3) a nucleic acid having a sequence with at least 95% homology to the sequence of the nucleic acid of 1) and encoding OTC;

[0010] 4) an mRNA comprising an OTC open reading frame;

[0011] 5) a codon-optimized mRNA encoding an OTC open reading frame;

[0012] 6) a nucleic acid partially or fully complementary to any one of 1) to 5);

[0013] 7) any one of the nucleic acids of 1) to 6) comprises a 5' untranslated region (UTR) sequence and a 3' untranslated region (UTR) sequence;

[0014] 8) any one of the nucleic acids of 1) to 6) comprises one or more modified nucleotides;

[0015] 9) any one of the nucleic acids of 1) to 6) is unmodified;

[0016] 10) any one of the nucleic acids of 1) to 6) comprises a circular RNA or a self- replicating RNA form.

[0017] In one embodiment of the present application, the modified nucleotides are selected from at least one of pseudouridine, 5-methoxyuridine, 5-methylcytidine, 2-thiouridine, N6-methyladenosine (m6A), N1-methyladenosine (mlA), 2'-O-methylation, N6-methyladenosine (m6A), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (5hmC), N4-methylcytidine (m4C), 7-methylguanosine (m7G), N2-methylguanosine (m2G), N1-methylpseudouridine, m2,7G, m2,2,7G, and Nm, etc.

[0018] In one embodiment of the present application, the modified nucleotides are at least one pseudouridine selected from 4-thiopseudouridine, 2-thiopseudouridine, 1-carboxymethylpseudouridine, 1-propynylpseudouridine, 1-taurinomethylpseudouridine, N1-methylpseudouridine, 4-thio-1-methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine, 2-thio-dihydrouridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thiopseudouridine, in particular N1-methylpseudouridine.

[0019] In one embodiment of the present application, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the nucleotides of the modified nucleotides are replaced with modified nucleotides. In one embodiment of the present application, the modified nucleotides comprise different nucleotide modifications in the same mRNA molecule.

[0020] In one embodiment of the present application, the mRNA further comprises a signal peptide coding sequence, a cap structure, and / or a tail structure.

[0021] In an embodiment of the present application, the mRNA can increase OTC gene expression by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.

[0022] In a second aspect of the present application, a DNA molecule is provided, which can be transcribed to obtain the mRNA.

[0023] In a third aspect of the present application, a recombinant expression vector is provided, which comprises the mRNA or the DNA molecule.

[0024] In an embodiment of the present application, the backbone vector of the recombinant expression vector is Puc57, pAAV-MCS, pcDNA3.1(+), pCMV-MCS, pEGFP-CTSB, or pLVX-PAX1.

[0025] In an embodiment of the present application, the promoter of the recombinant expression vector is Lac lactose operon, TAC promoter, TRC promoter, or T7 promoter.

[0026] In a fourth aspect of the present application, a transformant is provided, which comprises the mRNA, the DNA molecule, or the recombinant expression vector.

[0027] In an embodiment of the present application, the transformant is a prokaryotic cell and / or a eukaryotic cell, preferably an isolated mammalian cell, and more preferably an isolated human cell.

[0028] In a fifth aspect of the present application, a preparation is provided, which comprises the isolated mRNA, the DNA molecule, the recombinant expression vector, and / or the transformant, and a delivery agent.

[0029] In an embodiment of the present application, the delivery agent comprises a liposome nanoparticle (LNP), a liposome, a polymer, a micelle, a plasmid, a virus, or any combination thereof.

[0030] In an embodiment of the present application, the LNP is selected from a cationic lipid, a helper phospholipid, a sterol lipid, a polyethylene glycol (PEG)-modified lipid.

[0031] In an embodiment of the present application, the cationic lipid is preferably selected from one or more of 4-(N,N-dimethylamino)butanoic acid (dilinoleyl) methyl ester (DLin-MC3-DMA), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine (DLin-KC2-DMA), Bis((Z)-non-2-en-1-yl)-9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), MC3, DODAC, DDAB, DODMA, Dlin-DAC, C12-200, DODAP, HGT5000, HGT5001, XTC, ALNY-100, cKK-E12.

[0032] In an embodiment of the present application, the sterol lipid is preferably selected from one or more of cholesterol, cholesterol ester, sterol hormones, sterol vitamins, and phytosterols, more preferably one or more of cholesterol, cholesterol ester, and phytosterols, most preferably cholesterol.

[0033] In an embodiment of the present application, the helper phospholipid is preferably selected from one or more of DSPC, DOPC, DPPG, DOPS, and DOPE, more preferably DSPC and / or DOPS, most preferably DSPC.

[0034] In an embodiment of the present application, the PEG-modified lipid is preferably selected from one or more of DAG-PEG, DAA-PEG, DMG-PEG, Cer-PEG, and DSPE-PEG, more preferably PEG-DMG. In a preferred embodiment of the present application, the PEG has a relative molecular mass of 2000-5000, for example 2000, 3000, 4000, or 5000.

[0035] In an embodiment of the present application, the LNP is selected from MC3, DSPC, cholesterol, mPEG2000-DMG.

[0036] In an embodiment of the present application, the molar ratio of ionizable cationic lipid, helper lipid, sterol lipid, PEG lipid is (20-100):(5-20):(20-50):(0.5-5); preferably, the molar ratio is (30-60):(5-15):(30-40):(0.5-3), for example, in an embodiment of the present application, the molar ratio of ionizable cationic lipid, helper lipid, sterol lipid, PEG lipid is 50:10:38.5:1.5.

[0037] In an embodiment of the present application, the average particle size of the liposomal nanoparticle is 10-500 nm; preferably 80-120 nm.

[0038] In some embodiments, the mRNA, the DNA molecule, or the recombinant expression vector described above is complexed with a liposome nanoparticle to form a complex particle. In an embodiment of the present application, at least 95% of the mRNA, the DNA molecule, or the recombinant expression vector described above is encapsulated in the LNP. In an embodiment of the present application, the mRNA, the DNA molecule, or the recombinant expression vector described above is completely encapsulated in the LNP.

[0039] In one or more embodiments, the complex particle has a diameter of 80-120 nm.

[0040] In an embodiment of the present application, the mass ratio of the delivery agent to the mRNA, the DNA molecule, and / or the recombinant expression vector is (10-30):1, for example, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.

[0041] In an embodiment of the present application, the preparation further comprises a pharmaceutically acceptable excipient.

[0042] In an embodiment of the present application, the preparation comprises a lyophilized agent, an injection agent, or the like.

[0043] In an embodiment of the present application, the preparation is administered by intravenous, intramuscular, subcutaneous, or topical route.

[0044] In an embodiment of the present application, the preparation is an injection agent, and the administration is intravenous injection.

[0045] In an embodiment of the present application, the mRNA molecule is administered to the subject at a dose of 0.1-1.0 mg / kg, for example, at a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 10 mg / kg.

[0046] In an embodiment of the present application, the mRNA is administered to the subject for a duration of 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks. In a preferred embodiment, the mRNA of the present application is administered to the subject for a duration of 3 weeks.

[0047] In a sixth aspect of the present application, a pharmaceutical composition comprising the mRNA described above, the DNA molecule described above, the recombinant expression vector, the transformant, and / or the preparation is provided.

[0048] In an embodiment of the present application, the pharmaceutical composition further comprises a drug for treating and preventing liver fibrosis and / or a drug for treating HCC.

[0049] In some embodiments, the therapeutic drug for liver fibrosis includes, but is not limited to, FXR agonists such as obeticholic acid, thyroid hormone beta receptor (THR-beta) agonists such as Resmetirom, FGF 21 / 19 inhibitors, acetyl-CoA carboxylase (ACC) inhibitors, siRNA, shRNA, ASO RNA, etc.

[0050] In some embodiments, the therapeutic drug for HCC includes, but is not limited to, Sorafenib, Lenvatinib, Apatinib, Regorafenib, PD-1 mAb, PD-L1 mAb, CTLA4 mAb, TIGIT mAb, LAG3 mAb, VEGF mAb, TCR-T cells, CAR-T cells, CAR-macrophages, CAR-NK cells, siRNA, shRNA, ASO RNA, etc.

[0051] In one embodiment of the present application, the mRNA is administered in combination with a therapeutic and prophylactic drug for liver fibrosis and a therapeutic drug for HCC. The combination administration includes continuous administration in any order or at any time interval, so that two or more drugs exert their biological activity at the same time. Preferably, the combination administration produces a synergistic therapeutic effect.

[0052] In a seventh aspect of the present application, a preparation method of the above mRNA is provided, which comprises transcribing the above DNA molecule or expression vector to obtain the mRNA.

[0053] In an eighth aspect of the present application, a method for increasing the amount of OTC expression in cells is provided, which comprises introducing the above mRNA, the above DNA molecule and / or the above recombinant expression vector into the cells.

[0054] In a ninth aspect of the present application, the use of the above mRNA, the above DNA molecule, the above recombinant expression vector, the above composition, and the above preparation in the preparation of products for the following uses is provided:

[0055] 1) diagnosing, preventing or treating liver fibrosis;

[0056] 2) inhibiting or improving the progression of liver fibrosis;

[0057] 3) diagnosing or treating liver cancer;

[0058] 4) promoting the transdifferentiation of liver cancer cells into hepatocytes with partial liver function, restoring liver function, and prolonging the survival time of HCC patients;

[0059] 5) at least one of reducing the content of glutamic-pyruvic transaminase, reducing the content of glutamic oxalacetic transaminase, reducing the content of hydroxyproline, reducing the content of alpha-SMA or inhibiting the expression of alpha-SMA, reducing the content of Col1a1, Col1a2 and Acta2 or inhibiting the expression of Col1a1, Col1a2 and Acta2, reducing the content of AFP, reducing the concentration of blood ammonia;

[0060] 6) increasing the content or expression of Albumin, HNF4a, CYP1A2 and CYP3A4;

[0061] 7) at least one of increasing the ability of liver cells to polymerize glucose molecules, increasing the ability of liver cells to transport cholesterol, and increasing the reserve function of liver cells.

[0062] Compared with the prior art, the present application has the following beneficial effects:

[0063] (1) can treat liver fibrosis, achieve clinical liver fibrosis remission, and inhibit the progression of liver fibrosis;

[0064] (2) can promote the transdifferentiation of hepatocarcinoma cells into hepatocytes with partial liver function, restore liver function, and prolong the survival time of HCC patients;

[0065] (3) mRNA does not enter the nucleus, only expresses the target protein in the cytoplasm, and the mRNA itself does not have infectivity and can be degraded through normal cell pathways, having good safety. The protein translated from the mRNA in the inoculator's body has a high fidelity of spatial structure and post-translational modification such as glycosylation, and can efficiently achieve the complementation of missing proteins and restore their physiological functions;

[0066] (4) the delivery of OTC mRNA through liposomes has good targeting to liver cells, and can efficiently and specifically promote the expression of OTC mRNA in liver cells or hepatocarcinoma cells. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 Electrophoretograms of human and mouse OTC mRNA of the embodiments are shown.

[0068] Figure 2 Protein expression amount of mouse OTC mRNA of the embodiments in 293T cells and AML12 cells is shown.

[0069] Figure 3 Expression of human OTC mRNA of the embodiments in HepG2 cells is shown.

[0070] Figure 4 Pharmacodynamic evaluation of mouse OTC mRNA of the embodiments in inhibiting the progression of CCl4 mouse liver fibrosis model is shown.

[0071] Figure 5 Figure 6 shows the results of immunohistochemical staining of liver tissue from a CCl4 mouse liver fibrosis model treated with mouse OTC mRNA.

[0072] Figure 6 Figure 7 shows the results of immunohistochemical staining of liver cells from a CCl4 mouse liver fibrosis model treated with mouse OTC mRNA.

[0073] Figure 7 Figure 8 shows the results of Nessler's reagent staining of ammonia ion concentration in liver tissue from a CCl4 mouse liver fibrosis model treated with mouse OTC mRNA.

[0074] Figure 8 Figure 9 shows the results of Mdr2 - / - mouse spontaneous liver fibrosis model.

[0075] Figure 9 Figure 10 shows the results of Mdr2 - / - mouse spontaneous liver fibrosis model.

[0076] Figure 10 Figure 11 shows the results of immunohistochemical staining of liver cells from a Mdr2 - / - mouse spontaneous liver fibrosis model.

[0077] Figure 11 Figure 12 shows the results of Nessler's reagent staining of ammonia ion concentration in liver tissue from a Mdr2 - / - mouse spontaneous liver fibrosis model.

[0078] Figure 12 Figure 13 shows the results of promoting liver cell function recovery in liver cancer cells HepG2 treated with human OTC mRNA.

[0079] Figure 13 Figure 14 shows the results of promoting LDL uptake ability in liver cancer cells HepG2 treated with human OTC mRNA.

[0080] Figure 14 Figure 15 shows the results of inhibiting mouse liver orthotopic tumor progression efficacy treated with mouse OTC mRNA.

[0081] Figure 15 Figure 16 shows the results of Western-blot protein detection of liver cell function recovery in mouse liver orthotopic tumor treated with mouse OTC mRNA.

[0082] Figure 16Figure showing the results of immunohistochemical staining for liver function of mice with murine OTC mRNA restored hepatocyte function in orthotopic tumor in liver.

[0083] Figure 17 Figure showing the results of glycogen staining for liver function of mice with murine OTC mRNA restored hepatocyte function in orthotopic tumor in liver.

[0084] Figure 18 Figure 18A and 18B show the results of safety evaluation of the efficacy of murine OTC mRNA in inhibiting the progression of orthotopic tumor in liver of mice. 18A is the results of serum biochemical indicators; 18B is the results of HE detection. DETAILED DESCRIPTION

[0085] The technical solutions of the present application will be further described below in conjunction with specific examples. It should be understood that the following examples are only illustrative and explanatory of the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology implemented based on the above description of the present application is encompassed within the scope of the present application.

[0086] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0087] Technical terms

[0088] Messenger RNA (mRNA): refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA includes modified and unmodified RNA. An mRNA can contain one or more coding and non-coding regions. An mRNA can be purified from a natural source, produced using a recombinant expression system, or chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, an mRNA can comprise nucleotide analogs, e.g., analogs with chemically modified bases or sugars, backbone modifications, etc. Unless otherwise specified, mRNA sequences are presented in the 5' to 3' direction. In some embodiments, an mRNA comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleotide analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalating bases; modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5-N-phosphoramidate linkages).

[0089] 5' untranslated region (UTR) sequence: includes one or more elements that affect the stability or translation of the mRNA, such as an iron response element. In some embodiments, the 5' untranslated region can be about 50 to 500 nucleotides in length. In embodiments of the application, the 5' untranslated region sequence can comprise a 5' untranslated sequence of a gene selected from the group consisting of a beta-globin gene, a heat shock protein 70 gene, a centrin 2 gene, a hydroxysteroid (17-beta) dehydrogenase gene, and / or a KOZAK sequence, or a homolog, fragment or variant thereof.

[0090] 3' untranslated region (UTR): includes one or more of a polyadenylation signal, binding sites for proteins that affect the localization stability of the mRNA in the cell. In some embodiments, the 3' untranslated region can be 50 to 500 nucleotides in length or longer. In embodiments of the application, the 3' untranslated sequence can comprise a 3' untranslated sequence of a gene selected from the group consisting of an albumin gene, an alpha-globin gene, a beta-globin gene, a tyrosine hydroxylase gene, a heat shock protein 70 gene, a lipoxygenase gene, and a collagen alpha gene, or a homolog, fragment or variant thereof.

[0091] Cap structure: The mRNA of the application can comprise a 5' cap structure, a 5' cap analog, or a modified 5' cap structure. Preferably, the mRNA molecule can comprise a 5' cap structure selected from m7GpppN, ARCA Cap, or Capl. A preferred cap analog is m7G(5')ppp(5')G (m7G) or 3'-O-Me-m7G(5')ppp(5')G. Cap structures can be added using various art-recognized techniques. For example, a 5'-cap structure can be added co- transcriptionally in an in vitro transcription reaction of the RNA as appropriate.

[0092] Tail structure: Typically, a tail structure includes a poly(A) and / or poly(C) tail. It serves to protect the mRNA from exonuclease degradation. In certain embodiments, a long poly A tail can be added to the mRNA molecule, making the RNA more stable. Poly A tails can be added using various art-recognized techniques. For example, a long poly A tail can be added to a synthetic or in vitro transcribed RNA using a poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). The transcription vector can also encode a long poly A tail. Additionally, a poly A tail can be added by direct transcription from a PCR product. Poly A can also be ligated to the 3' end of the sense RNA using an RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd edition).

[0093] Delivery: includes local delivery and systemic delivery. For example, delivery of mRNA includes the case where mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as "local delivery"), and the case where mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum), and is distributed systemically and taken up by other tissues (also referred to as "systemic delivery").

[0094] Encapsulation: refers to the process of confining individual mRNA molecules within a nanoparticle.

[0095] Expression: refers to the translation of mRNA into a polypeptide, the assembly of a polypeptide into a complete protein (e.g., enzyme), and / or post-translational modification of a polypeptide or completely assembled protein (e.g., enzyme). In this patent application, the terms "expression" and "production" are used interchangeably.

[0096] Modified: refers to the altered state or structure of an mRNA molecule. An mRNA molecule can be modified in a variety of ways, including chemically, structurally, and functionally. In some examples, the mRNA molecules of the present application are modified by the introduction of non-natural nucleosides and / or nucleotides, e.g., that are related to the natural ribonucleotides A, U, G, and C.

[0097] Pharmaceutically acceptable: refers to a substance that is, within the scope of sound medical judgment, appropriate for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0098] Pharmaceutically acceptable excipient: refers to any ingredient that has been reasonably assessed for safety and is included in a pharmaceutical formulation (e.g., a vehicle that can suspend or dissolve an active compound) and has substantially non-toxic and non-inflammatory properties in patients. These excipients have important functions in addition to serving as carriers and excipients for drugs, such as improving drug stability, improving drug release and absorption, etc. The excipients in the present application can include, for example: anti-adhesion agents, antioxidants, binders, coatings, tabletting aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium hydrogen phosphate (dibasic), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, nipagin methyl ester, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, nipagin propyl ester, retinol palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium carboxymethyl starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0099] Example 1 Preparation of OTC-mRNA

[0100] 1. Design of OTC mRNA sequence

[0101] The coding sequence (CDS) region is the protein coding region of mRNA, which is crucial for the translation and stability of mRNA. The optimization of CDS mainly focuses on codon optimization. The optimization scheme is mainly based on two parameters, namely Codon Adaption Index (CAI) and Minimum Free Energy (MFE). The CAI parameter is related to the codon preference. In an organism, any amino acid corresponds to multiple different codons, but in the translation process, the frequency of using one codon is dominant. When optimizing the sequence with CAI as the parameter, the preferred codon will be used as much as possible, thereby increasing the translation rate and accelerating protein synthesis. The MFE parameter is related to the stability of mRNA secondary structure. The lower the MFE, the more stable the secondary structure of the mRNA molecule, and the longer the half-life. Based on MFE, the optimization of codon will improve the in vivo stability of mRNA molecules and ensure the relatively long-term and continuous expression of antigens. The optimized OTC mRNA sequence includes 2 murine sequences and 1 human sequence as shown below:

[0102] SEQ ID NO: 1 murine OTC-01:

[0103]

[0104]

[0105] SEQ ID NO: 2 murine OTC-02:

[0106]

[0107] SEQ ID NO: 3 human OTC sequence:

[0108]

[0109]

[0110] 2. Preparation of OTC-mRNA

[0111] 1) Whole gene synthesis of template plasmid

[0112] After the ORF (open reading frame) sequence of OTC is concatenated with the T7 promoter sequence, 5'UTR sequence, 3'UTR sequence and polyA sequence, whole gene synthesis is performed with Puc57 as the carrier to obtain the template plasmid.

[0113] 2) PCR to obtain transcription template DNA sequence

[0114] Using the linearized template plasmid as the template, using polyT long primer, high-fidelity DNA polymerase (purchased from Thermo Scientific Phusion), dNTP, etc. raw materials, using a suitable program on the PCR instrument, the transcription template DNA (IVT template) can be obtained.

[0115] 3) Preparation of mRNA by in vitro transcription reaction (using 40 μL reaction system as an example)

[0116] The prepared IVT template is mixed with T7 RNA polymerase (purchased from NEB), rNTPs mononucleotides, etc. raw materials according to the recommended ratio, co-transcription capping method is adopted, and the transcription reaction is carried out at 37°C for 2 hours. After the transcription reaction is completed, the IVT template is digested using DNAase (purchased from Novozymes) to reduce the risk brought by residual DNA template.

[0117] 4) Purification

[0118] The mRNA from the IVT reaction was purified using an RNA purification kit (NEB). The purified mRNA was dissolved in TE buffer and ready for subsequent formulation coating. Electrophoresis results ( Figure 1 The results showed that the mRNA purity was good.

[0119] 3. OTC mRNA sequence transfection into HEK-293T, AML12, and HepG2 cells was used to verify OTC protein expression levels.

[0120] 293T, AML12, or HepG2 cells were revived and seeded into 12-well plates when they reached 80-100% confluency. After 12 hours, when the cells reached 70% confluency, the culture medium was replaced with serum-free medium, and mRNA was transfected into 293T, AML-12, or HepG2 cells using a Lipofectamine transfection kit (purchased from Polyplus). Cells with liposomes alone were used as negative controls. Detection was started 48 hours after transfection. For time-series assays, cells were collected at 48h, 72h, and 96h, and an appropriate amount of lysis buffer was added. The cells were gently mixed by pipetting, incubated on ice for 30 minutes, centrifuged at 15000 rpm for 15 minutes, and the supernatant was collected. The expression abundance of the target protein OTC was determined using Western blot.

[0121] Experimental results:

[0122] High levels of OTC expression were detected intracellularly in both HEK-293T and AML-12 cells after transfection with mouse OTC-mRNA-1 and 2 sequences, showing a dose-dependent upregulation. Figure 2 (A, 2C) OTC expression levels peaked at 48 hours and began to decline after 72 hours. Figure 2 B, 2D), among which OTC-2-Ψ modification resulted in the highest abundance of intracellular OTC ( Figure 2 Therefore, we chose the OTC-2-Ψ sequence for subsequent experiments. Simultaneously, we designed human OTC sequences using the OTC-2-Ψ modification method, and OTC expression was dose-dependently upregulated after transfection of HepG2 cells. Figure 3 A) OTC expression levels peaked at 48 hours and began to decline after 72 hours. Figure 3 B).

[0123] 4. Detection of mRNA encapsulated by liposome nanoparticles and their particle size

[0124] Preparation of lipid solution: Dissolve MC3:DSPC:cholesterol:mPEG2000-DMG in ethanol solution at a molar ratio of 50:10:38.5:1.5;

[0125] mRNA solution preparation: a certain mass of OTC mRNA was dissolved in 20 mM citric acid buffer solution with pH = 4.0;

[0126] Preparation of lipid nanoparticles: 1 mL of OTC mRNA and 3 mL of lipid solution were respectively taken by a syringe and inserted into the microfluidic chip, and the parameters were set as follows: Volume: 4.0 mL; Flowrate ratio: 3:1, Total flow rate: 18 mL / min, and the lipid nanoparticle solution was obtained by mixing;

[0127] Solution replacement: the lipid nanoparticle solution was added into an ultrafiltration tube for centrifugal ultrafiltration, and phosphate buffer was used for multiple replacement to obtain the finished product.

[0128] Particle size detection: the particle size and shape were analyzed by a laser particle size and shape analyzer using laser diffraction method and dynamic image method, and the particle size was 70-88 nm.

[0129] Example 2: Construction of a liver fibrosis mouse model and LNP-OTC mRNA treatment strategy

[0130] 1. Construction of a liver fibrosis mouse model

[0131] 1) CCL4 mouse liver fibrosis model

[0132] Olive oil was configured with 20% CCl4, and 7-week-old C57BL / 6J mice were given intraperitoneal injection, with a dose of 1 mL / kg, twice a week, and the mice developed liver fibrosis symptoms after 6 weeks.

[0133] 2) Mdr2 - / - Spontaneous liver fibrosis model in mice

[0134] When Mdr2 is deleted, the concentration of phospholipids in the bile duct decreases, and the bile components lacking phospholipids can cause damage to the bile duct, gallstone deposition, induce inflammation and further cause liver fibrosis. Mdr2 knockout mice develop hepatocyte damage, vascular dilation and ductal hyperplasia phenotype 2-3 weeks after birth, and liver fibrosis symptoms appear after 8-9 weeks. The present application selects 12-week-old Mdr2 - / - Mice for OTC mRNA treatment of liver fibrosis efficacy evaluation.

[0135] 2. LNP-OTC mRNA treatment strategy

[0136] 1) CCL4 mouse liver fibrosis model

[0137] After three weeks of CCL4 modeling, the experimental group was injected with LNP-OTC mRNA drugs at a dose of 0.3 mg / kg through the tail vein, and the blank control group included no injection of any drugs, injection of the same volume of NaCl, and injection of the same volume of LNP-mRNA (GFP), with an interval of 3 days for tail vein injection, a total of 6 times. After the last administration, the eyeball was removed to collect blood, and the liver was collected and stored in liquid nitrogen / -80°C refrigerator. The treatment process is shown in FIG. 1A. Figure 4 A.

[0138] 2) Mdr2 - / - Spontaneous liver fibrosis model in mice

[0139] Twelve-week-old Mdr2 - / - Mice were used for the evaluation of the efficacy of OTC mRNA treatment of liver fibrosis, and the experimental group was injected with LNP-OTC mRNA drugs at a dose of 0.3 mg / kg through the tail vein, and the blank control group included no injection of any drugs, injection of the same volume of NaCl, and injection of the same volume of LNP-mRNA (GFP), with an interval of 3 days for tail vein injection, a total of 6 times. After the last administration, the eyeball was removed to collect blood, and the liver was collected and stored in liquid nitrogen / -80°C refrigerator. The treatment process is shown in FIG. 1A. Figure 8 A.

[0140] 3, Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) detection (Nanjing Jiancheng Bioengineering Institute, C009-2-1, C010-2-1)

[0141] Serum sample collection: The collected mouse blood was centrifuged at 2500 rpm, 4°C for 30 min, and then the upper serum was taken into a new PCR tube for AST and ALT detection or stored in a-80°C refrigerator.

[0142] The matrix solution was preheated in a 37°C biochemical incubator for 30 min.

[0143] AST and ALT sample wells were prepared according to the following Table 1:

[0144] Table 1 AST and ALT sample addition table

[0145]

[0146] AST and ALT standard curve preparation was performed according to the following Table 2:

[0147] Table 2 Standard curve addition

[0148]

[0149]

[0150] Note: Determination of holes, control hole standard curve hole every suction 1 sample, the gun head into the hole plate bottom matrix liquid, repeatedly suction and hit the mixture, do not produce bubbles.

[0151] Gently shake the 96-well plate, mix the liquid in the hole, and place it at room temperature for 15 min, then use the multifunctional enzyme marker to measure the absorbance value of each hole at 510 nm, draw the standard curve according to the absorbance and concentration of the standard, and substitute the absolute OD value (absolute OD value = determination hole OD value-control hole OD value) into the standard curve to obtain the corresponding ALT or AST activity unit. The results are as follows Figure 4 B, 4C or 8B, 8C.

[0152] 4. Hydroxyproline detection (Elabscience, E-BC-K062-S)

[0153] Hydrolysis of tissue samples: accurately weigh 0.1 g of liver tissue sample, cut into small pieces and put into a glass test tube, add 6 mol / L hydrochloric acid solution 1 mL, cover and seal, hydrolysis at 95℃ for 6h.

[0154] According to the reagent instruction, the specific steps are as follows:

[0155] ① Standard tube: take 400 μL of different concentration standard, respectively added to 2 mL EP tube. Measuring tube: take 400 μL of sample to be tested, added to 2 mL EP tube. ② Add 200 μL of reagent one working solution to each tube of step ①. ③ Mix well, room temperature for 15 min. ④ Add 400 μL of reagent four working solution to each tube of step ③. ⑤ Mix well, 60℃ water bath for 15 min. ⑥ Cool with running water, each tube is added to 0.5 cm light path quartz cuvette, double distilled water zero. ⑦ Use ultraviolet-visible spectrophotometer to measure the OD value of each tube at 558 nm wavelength. The results are as follows Figure 4 D or 8D.

[0156] From the results of steps 3 and 4, it can be seen that by establishing CCL4 induced liver fibrosis mouse model and MDR2 - / - Pharmacodynamic evaluation in spontaneous liver fibrosis model of mice, it was found that 0.3 mg / kg LNP-OTC mRNA significantly inhibited the progression of liver fibrosis in mice, and significantly reduced the content of serum liver damage indicators ALT, AST and hydroxyproline.

[0157] 5. Collagen-related protein detection in liver tissue

[0158] The liver tissue blocks stored in liquid nitrogen and -80°C refrigerator were cut into green bean size, and then the tissue blocks were placed in 1.5 mL EP tubes containing steel balls, 0.75 mL Ripa lysis buffer was added for lysis, and the tissue grinder was used for 65 Hz oscillation for 90-120 s; 12000 rpm, 4°C centrifugation for 10 min, and the supernatant was taken for the next experiment or stored in -80°C refrigerator. Western Blot was used to detect the contents of liver fibrosis related proteins a-SMA and COL1A1. The results are as follows Figure 4 E or 8E.

[0159] Protein detection showed that LNP-OTC mRNA significantly inhibited the expression of collagen-related proteins COL1A1 and a-SMA in liver tissues of two fibrosis models.

[0160] 6. Expression of collagen-related genes Col1a1, Col1a2 and Acta2 in liver tissues

[0161] 1) Tissue RNA extraction

[0162] ① The tissue blocks stored in -80°C refrigerator / liquid nitrogen were cut into green bean size, and then the tissue blocks were placed in 1.5 mL EP tubes containing steel balls, 1 mL Trizol (TAKARA, 9108) was added for lysis, and the tissue grinder was used for 65 Hz oscillation for 90-120 s; ② The upper aqueous phase was taken into a pre-frozen EP tube, and the same volume of isopropanol was added, and the EP tube was shaken back and forth vigorously for 35-40 times; 10 min on ice, 4°C, 12000 rpm centrifugation for 10 min; ③ The supernatant was carefully discarded, 1 mL 75% DEPC alcohol was added to blow up the precipitate, 4°C, 12000 rpm centrifugation for 3 min (repeat 2 times); the supernatant was carefully discarded, and the residual alcohol was absorbed after instantaneous separation, and the precipitate was placed on ice and volatilized for 15 min; ④ According to the size of the precipitate, 15-20 μL RNase Free Water was added, and the RNA was dissolved by tapping the bottom of the tube; ⑤ After dissolution, 1.0 μL was taken for nucleic acid quantification, and the rest was used for Real-time PCR and stored in -80°C refrigerator.

[0163] 2) Real-time fluorescent quantitative polymerase chain reaction detection

[0164] ① Remove genomic DNA (TAKARA, RR047A)

[0165] Table 3 gDNA removal reaction system

[0166]

[0167] According to the reaction system of table 3, the reagents, RNA and water, were added in sequence, mixed, and then centrifuged instantly. The sample was placed in a PCR instrument and reacted at 42℃ for 2min. When the temperature dropped to 4℃, the reaction sample was taken out.

[0168] ②Reverse transcription (Takara, RR047A)

[0169] Table 4 Reverse transcription reaction system

[0170]

[0171] According to the reaction system of table 4, the reagents were added in sequence, mixed, and then centrifuged instantly. The sample was placed in a PCR instrument and reacted at 37℃ for 15min, 85℃ for 5s, and then the temperature dropped to 4℃. When the reaction sample was taken out, the sample obtained at this time was cDNA.

[0172] ③Real-time fluorescence quantitative PCR (Real-time PCR) (Vazyme, Q331-02)

[0173] Table 5 RT-qPCR reaction system

[0174]

[0175]

[0176] According to the reaction system of table 5, the reagents were prepared into a premix solution, 18μL of which was added to a Real-time PCR eight-tube tube. Finally, the sample cDNA was added in sequence (two duplicate holes were set for each sample), and then centrifuged instantly and placed in a PCR instrument. The program is shown in table 6:

[0177] Table 6 RT-qPCR reaction program

[0178]

[0179] ④Data processing (ΔΔCt method)

[0180] The data was exported and summarized. The Ct value of the target gene of each sample was subtracted from the Ct value of the internal reference gene: ΔCt 样本 =Ct 样本 -Ct 内参 . The average ΔCt value of the control group was taken as ΔCt 对照 : ΔΔCt = ΔCt 样本 -ΔCt 对照 . 2 -ΔΔCt was calculated, and the data was analyzed and plotted using GraphPad prism 9.0.

[0181] The results are as follows: Figure 4F or 8F. Transcriptional level detection found that LNP-OTC mRNA significantly inhibited the expression of collagen-related genes Col1a1, Col1a2 and Acta2 in liver tissues of two fibrosis models.

[0182] 7. HE staining

[0183] (1) Paraffin section deparaffinization: put the section into xylene I for 10 min; xylene II for 10 min; anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min; 95% alcohol for 5 min; 90% alcohol for 5 min; 80% alcohol for 5 min; 70% alcohol for 5 min; distilled water for 5 min.(2) Stain the nucleus with hematoxylin: put the section into Harris hematoxylin for 3-8 min, and wash with tap water.(3) Stain the cytoplasm with eosin: put the section into eosin staining solution for 1-3 min, and wash with tap water.(4) Dehydration and mounting: put the section into 95% alcohol I for 5 min; 95% alcohol II for 5 min; anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min; xylene I for 5 min; xylene II for 5 min; after dehydration and transparency, take the section out of xylene and dry slightly, and mount with neutral balsam.(5) Microscope examination, image collection and analysis.

[0184] Results are shown in Table 1. Figure 5 or Figure 9 Results show that histopathological staining results show that LNP-OTC mRNA significantly inhibits the damage of liver tissues of two fibrosis models.

[0185] 8. Immunohistochemical detection of collagen-related protein expression and distribution in liver tissues

[0186] ①Fixation: The fresh tissue was fixed with 4% paraformaldehyde. The tissue was cut into blocks with flat sections and then placed in a box with 4% paraformaldehyde for further fixation; ②Block: After dehydration treatment, the fixed tissue block was embedded with paraffin; ③Sectioning, spreading and picking up: After sectioning with a paraffin microtome (thickness of 4), the sections were placed in a 45°C hot water bath until they were flat and then picked up with a glass slide treated with polylysine and labeled, and then placed in a 60°C oven for overnight drying; ④Dewaxing and rehydration: 60°C xylene I was used for dewaxing for 30 min, 10 min of room temperature xylene II, and then 3 min of immersion in alcohol solutions with concentrations of 100%, 95%, 85%, 75%, and 50%, respectively, followed by 3 min of rinsing in double-distilled water I and II; ⑤Antigen retrieval: A beaker with a capacity of 1 L was filled with an appropriate amount of 10 mM citrate buffer solution with a pH of 6.0 or basic Tris-EDTA buffer solution with a pH of 9.0, and the sections were placed in the buffer solution, completely immersed, covered with a layer of plastic wrap (to prevent liquid evaporation), and punctured with several air holes in the plastic wrap to prevent violent boiling. A microwave oven was used for high-heat heating until boiling began (about 10 min), and then the medium-heat heating was continued for 20 min. After the sections were taken out, they were naturally cooled to room temperature; ⑥Permeabilization: The sections were placed in a PBS solution with a final concentration of 0.3% Triton X-100 for permeabilization for 15 min, and then washed with PBS for 5 min; ⑦Blocking: The sections were covered with 10% goat serum for blocking for 1 h; primary antibody incubation: the blocking solution was removed, 50 μL of the primary antibody prepared with 10% goat serum was added, and the sections were placed in a wet box for overnight incubation in a 4°C refrigerator; ⑧Rewarming: the next day, the wet box with the sections was placed in a 37°C incubator for rewarming for 45 min, and then washed with PBST for 3 times, 5 min each time; ⑨Blocking endogenous peroxidase: after 20 min of immersion in a methanol solution containing 0.3% H2O2, the sections were immediately taken out and washed with PBST for 3 times, 5 min each time; ⑩Secondary antibody incubation: the secondary antibody was added, the sections were placed in a wet box, and incubated at room temperature for 20 min, and then washed with PBST for 3 times, 5 min each time; Color development: DAB color development for 1-3 min, and then washed with pure water; Nuclear staining: hematoxylin staining for 3 min, and then washed with tap water to remove residual purple color; Differentiation: 1% hydrochloric acid alcohol differentiation for 10-30 s; Counterstaining: 1% ammonia water counterstaining for 2 min, and then placed in pure water; Dehydration: sequentially placed in 50%, 75%, 85%, 95%, and 100% I and 100% II alcohol solutions for 2 min; Permeabilization: xylene I and xylene II preheated in a water bath were used for permeabilization for 3 min each; Mounting: neutral resin was added, and then covered with a cover glass, and air-dried. The expression and distribution of α-SMA and OTC proteins were detected by the above method.

[0187] Results are shown in Figure 5 , Figure 6 or Figure 9 , Figure 10 . Results show that immunohistochemistry staining results show that LNP-OTC mRNA significantly increased OTC expression in liver tissue Figure 6 , Figure 10 and significantly inhibited the expression of a-SMA in liver tissue Figure 5 , Figure 9 .

[0188] 9. Masson staining (Solarbio, G1340)

[0189] ①Paraffin section dehydration (same steps as HE); ②Stained with prepared Weigert iron hematoxylin staining solution for 5-10 min; ③Acetic acid differentiation for 5-15 s; ④Masson blue solution counterstaining; ⑤Lilac red staining solution for 5-10 min; ⑥Weak acid working solution was prepared according to the ratio of distilled water: weak acid solution = 2:1, and washed with weak acid working solution for 1 min; ⑦Phosphomolybdate solution for 1-2 min, and prepared weak acid working solution for 1 min; ⑧Put into aniline blue staining solution for 1-2 min, and prepared weak acid working solution for 1 min; ⑨95% ethanol rapid dehydration for 2-3 s, and anhydrous ethanol dehydration for 3 times, each time for 5-10 s; ⑩Xylene transparency for 3 times, each time for 1-2 min, and neutral resin fixation.

[0190] Results are shown in Figure 5 or Figure 9 . Results show that Masson staining results show that LNP-OTC mRNA significantly inhibits the expression of collagen in liver tissue.

[0191] 10. Nessler's reagent staining

[0192] Paraffin-embedded glass slides were subjected to ammonia staining with Nessler's reagent (Merck, 109028) according to the following protocol: The slides were deparaffinized in xylene and dehydrated in an alcohol gradient (same steps as HE). Then the slides were placed in a humid chamber with distilled water to avoid dehydration during staining. Each section was incubated with 100 mL of Nessler's reagent for 5 min, and washed with sterile distilled water for 10 s. The slides were counterstained with Mayer's hematoxylin, rinsed with tap water, briefly dehydrated, and then cleared with histoclear (two changes of 20 s). This Nessler's reagent solution turns deep yellow in the presence of ammonia. At high concentrations of ammonia, this reaction leads to the formation of a brown precipitate.

[0193] Results are shown in Figure 7 or Figure 11As shown, the nessler reagent staining results show that LNP-OTC mRNA significantly reduces the ammonia ion concentration in liver tissue. The above results prove that LNP-OTC mRNA is an effective drug for relieving liver fibrosis.

[0194] Example 3 Function detection of human OTC mRNA sequence transfected HCC cells inducing hepatocyte-like cell differentiation

[0195] 1. Cell transfection

[0196] The same as step 3 of example 1.

[0197] Specifically, the HepG2 cells were resuscitated, and when they reached 80-100% density, the cells were inoculated in a 6-well plate. After 12h, when the cells grew to 70% density, the culture medium was replaced with serum-free culture medium, and human OTC mRNA was transfected into HepG2 cells by transfection reagent kit Lipofectamine (jetMES reagent, Polyplus). The liposome transfected cells were used as a blank control, and the detection was started after 48h of transfection. The samples were collected at 48h, 72h and 96h for detection. The results are shown in Figure 3

[0198] 2. Detection of hepatocyte function proteins

[0199] Pre-cool 1xPBS for two times, add appropriate amount of cell lysis solution, lyse on ice for 10min, scrape the cells with a spatula, then transfer the lysis solution to a 1.5mL EP tube and lyse on ice for 10min, vortex 2-3 times; BCA method for protein quantification; HNF4α, CYP1A2 and Transferrin were detected by Western Blot.

[0200] The results are shown in Figure 12 A. The results show that LNP-OTC mRNA can promote the expression of hepatocyte function proteins Transferrin, CYP1A2 and HNF-4α;

[0201] 3. Glycogen staining

[0202] ​​Cell plating (six-well plate) 50000 cells per well, 24h later, use Solarbio glycogen PAS staining kit for staining, the specific staining steps are as follows: discard the culture medium, wash with PBS for 3 times and absorb the residual liquid in the hole; add 1 mL oxidant to each hole for 5-10 min, discard the oxidant after treatment, wash with PBS for 3 times and absorb the residual liquid in the hole; add 1 mL Schiff's staining solution to each hole for 10-15 min, discard the staining solution after treatment, wash with PBS for 3 times, and keep about 1 mL PBS per hole, observe the glycogen production in the cells under a microscope. The results are shown in Figure 12 B. The results show that LNP-OTC mRNA promotes the polymerization of glucose molecules in hepatocytes in a dose-dependent manner.

[0203] 4. Indocyanine green (ICG) uptake experiment

[0204] Cell plating (six-well plate) 50000 cells per well; 24h later, add indocyanine green, dilute to 500mg / mL with cell culture medium; incubate at 37°C for 2h; after incubation, absorb the culture medium containing indocyanine green, wash with PBS until there is no obvious color; keep about 1 mL PBS per hole, observe the indocyanine green uptake in the cells under a microscope. The results are shown in Figure 12 B. The results show that LNP-OTC mRNA promotes the uptake of indocyanine green in hepatocytes in a dose-dependent manner.

[0205] 5. LDL uptake experiment

[0206] Cell plating (96-well plate): 10000 cells per well, 24h later, add Human DiI-Ac-LDL, dilute to 25μg / mL with cell culture medium; incubate at 37°C for 4h; after incubation, absorb the culture medium containing Human DiI-Ac-LDL, wash with PBS for 3 times; observe the uptake amount in the cells under a fluorescence microscope. The results are shown in Figure 13 B. The results show that LNP-OTC mRNA promotes the uptake of LDL in hepatocytes in a dose-dependent manner.

[0207] The glycogen staining reaction of hepatocytes to the polymerization of glucose molecules, the indocyanine green uptake reaction of hepatocytes to the reserve function, and the LDL uptake reaction of hepatocytes to the transport of cholesterol ability, the above results prove that LNP-OTC mRNA promotes HepG2 cells to restore hepatocyte function. The in vitro experiment further proves that LNP-OTC mRNA can promote hepatocarcinoma cells to differentiate into cells with partial hepatocyte function.

[0208] Example 4 Construction of HCC mouse model and LNP-OTC mRNA treatment strategy

[0209] 1. Construction of HCC mouse model

[0210] 20 μg PT3-EF1a-c-Myc, 20 μg pT3-EF1a-NRasV12 and 1.6 μg pCMV-SB100x plasmids were dissolved in 2 mL sterile saline, and injected into 7-week-old C57BL / 6J mice by high-pressure hydrodynamic injection (7-8 s) through the tail vein. After 6-7 weeks, the mice developed orthotopic tumors in the liver.

[0211] 2. LNP-OTC mRNA treatment strategy

[0212] The experimental group was injected with LNP-OTC mRNA drugs at doses of 0.5 mg / kg and 1 mg / kg through the tail vein, and the blank control group was injected with the same volume of LNP. The injection was performed once every 3 days, and a total of 7 times. After the last administration, the eyeball was removed for blood collection, and the liver was collected and stored in liquid nitrogen and a -80°C refrigerator. Part of the liver was fixed in 4% paraformaldehyde. The treatment process is shown in Figure 14 A. The size of the liver is shown in Figure 14 B. The results show that the liver of the LNP-OTC mRNA drug group is significantly smaller, and is time- and dose-dependent.

[0213] 3. Serum ALT and AST detection

[0214] The experimental steps are the same as those in Example 2, step 3.

[0215] The results are shown in Figure 14 C, 14D. The results show that the liver damage indicators ALT and AST are significantly decreased.

[0216] 4. Serum AFP detection (Elabscience, Mouse αFP (Alpha-Fetoprotein) ELISA Kit, E-EL-M2405c)

[0217] ①Prepare standard solution: centrifuge the standard solution at 10000g for 1 min, add 1 mL of sample dilution to the standard solution, invert to dissolve the powder, stand for 10 min, and mix gently; ②Dilute the standard solution by 5 times: take 7 1.5 EP tubes, add 500 μL of standard solution to each tube, and dilute the standard solution from 10 ng / mL to 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.63 ng / mL, 0.31 ng / mL, 0.16 ng / mL, and 0 ng / mL; ③Dilute the sample to be tested by 5 times; ④Take the kit out of the refrigerator and stand at room temperature for 20 min, add 100 μL of standard solution and 100 μL of sample to be tested to the corresponding wells, cover the enzyme-labeled plate with the film, and incubate the enzyme-labeled plate in a biochemical incubator at 37°C for 90 min; ⑤Discard the liquid in the wells, add 100 μL of biotinylated antibody working solution to each well, cover the enzyme-labeled plate with the film, and incubate in a biochemical incubator at 37°C for 1 h; ⑥Discard the liquid in the wells, absorb the liquid on a clean absorbent paper, add 350 μL of washing solution to each well, soak for 1 min, and discard the liquid; repeat step 3 for 3 times; ⑦Add 100 μL of enzyme conjugate working solution to each well, cover with the film, and incubate in a biochemical incubator at 37°C for 30 min; ⑧Discard the liquid in the wells, add 350 μL of washing solution to each well, and discard the liquid; repeat step 5 for 5 times; ⑨Add 90 μL of substrate solution to each well, cover with the film, and incubate in a biochemical incubator at 37°C for 20 min; ⑩Add 50 μL of stop solution to each well, and immediately measure the absorbance value of each well at 450 nm wavelength using an enzyme-labeled instrument.

[0218] The results are shown in Table 1. Figure 14 E. The results show that the serum marker protein AFP of liver cancer is significantly decreased.

[0219] 5. Serum Ammonia detection (Elabscience, Blood Ammonia Colorimetric Assay Kit, E-BC-K145-M)

[0220] Serum sample: direct determination (note: avoid red blood cell hemolysis; store the sample at 2-8°C for 2-4 h, or at -20°C for 24 h; seal the sample to avoid ammonia overflow.)

[0221] According to the reagent instruction, the detection is carried out, and the specific steps are as follows:

[0222] ① Standard tube: take 100 μL of each concentration of standard sample application liquid and add to a 1.5 mL EP tube; measurement tube: take 100 μL of sample to be measured and add to a 1.5 mL EP tube; ② To each tube of step ①, add 300 μL of reagent one, vortex to mix, centrifuge at 1100 x g for 10 min. (After centrifugation, the color development reaction must be performed within 20 min); ③ Take 40 μL of supernatant from each tube of step ② and add to an enzyme-labeled plate well; ④ To each enzyme-labeled plate well of step ③, add 120 μL of reagent two and 120 μL of reagent three in turn. (Reagent two and reagent three cannot be mixed and then used); ⑤ Shake the plate on the enzyme marker for 5 s, incubate at 37°C for 25 min, and measure the OD value at 635 nm on the enzyme marker.

[0223] Results are shown in Figure 14 F. The results show that the blood ammonia concentration is significantly reduced.

[0224] 6. Hepatocyte function protein detection

[0225] Take the tissue block stored in liquid nitrogen and stored in a -80°C refrigerator, cut into a size of a green bean, and place the tissue block in a 1.5 mL EP tube containing a steel ball, add 0.75 mL of Ripa lysis buffer for lysis, and place on a tissue grinder at 65 Hz for 90-120 s; centrifuge at 12000 rpm at 4°C for 10 min, and take the supernatant for the next step experiment or store in a -80°C refrigerator. HNF-4α, CYP1A2, CYP3A4, Albumin, Transferrin and AFP are detected by Western Blot.

[0226] Results are shown in Figure 15 WB detection shows that the expression of liver function related proteins Albumin, HNF4α, CYP1A2 and CYP3A4 in the LNP-OTC mRNA treatment group is significantly increased, and the expression of AFP is significantly reduced.

[0227] 7. Immunohistochemical detection of hepatocyte function protein expression and distribution

[0228] The experimental steps are the same as those in Example 2, step 8, and the expression and distribution of tumor proliferation index ki67, liver function related proteins HNF4α, CYP1A2 and CYP3A4 are detected.

[0229] Results are shown in Figure 16 Immunohistochemical detection shows that the tumor proliferation index Ki67 in the LNP-OTC mRNA treatment group is significantly reduced, and the expression of liver function related proteins HNF4α, CYP1A2 and CYP3A4 is significantly increased.

[0230] Serum biochemical and pathological tests showed that LNP-OTC mRNA did not cause significant damage. The above results prove that LNP-OTC mRNA can significantly inhibit the progression of hepatocellular carcinoma, significantly promote the differentiation of hepatocellular carcinoma cells into partial hepatocyte functional cells, and restore partial liver function.

[0231] 8. Tissue glycogen staining (Solarbio, G1286)

[0232] ① 60℃ oven baking, until the wax block melts; ② Put into the preheated 60℃ xylene I for 30min, and room temperature xylene II for 10min; ③ Soak in alcohol solutions with concentrations of 100%, 95%, 85%, 75%, and 50% for 3min respectively, and then rinse in pure water I and II for 3min; ④ Dry with water, draw a wax circle with a wax pen, and then treat with an oxidizing agent for about 10min, and then rinse with running water for three times; ⑤ Dry the excess water on the slice, and then add Schiff's dye, and then dye for about 15min, and then rinse with running water for three times; ⑥ Hematoxylin re-stain for 10-15s, rinse the excess color with pure water, differentiate with HCl-alcohol for 10-30s, return to blue with ammonia water for 1min, and then put into pure water; ⑦ Soak in alcohol solutions with concentrations of 50%, 75%, 85%, 95%, and 100% I, and 100% II for 2min respectively for dehydration, and then put into xylene for 15min; ⑧ Use neutral resin to mount the slice, and then observe under a microscope.

[0233] The results are shown in Table 1. Figure 17 The results of tissue glycogen staining show that LNP-OTC mRNA significantly increases the glycogen content in the tissue, and the above results prove that LNP-OTC mRNA is an effective drug for promoting the differentiation of HCC cells.

[0234] 9. Safety evaluation of LNP-OTC mRNA tail vein injection

[0235] At the end of the experiment, the mice were taken blood from the eye orbit, and the mouse serum was collected for detection by a full-automatic biochemical analyzer (Siemens, Dimension Xpand Plus). The detection indexes include alkaline phosphatase (ALP), albumin (ALB), glutamyl transpeptidase (GGT), total cholesterol (CHOL), urea nitrogen (BUN), total protein (TP), and total bilirubin (TBI) and the like.

[0236] At the end of the experiment, the mice were taken heart, liver, spleen, lung, and kidney for HE staining. The experimental steps are the same as those in step 7 of Example 2.

[0237] The results are shown in Table 1. Figure 18As shown, the serum biochemical indicators and HE detection results showed that tail vein injection of LNP-OTC mRNA had no significant damage to the main organs of mice, and the above results proved the safety of tail vein injection of LNP-OTC mRNA.

[0238] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present application, and they should all be covered in the scope of the claims of the present application.

Claims

1. An mRNA, characterized in that, The mRNA comprises at least one of the following 1) - 10): 1) a nucleic acid having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1-3; 2) a nucleic acid in which one or more nucleotides are substituted, deleted, or added in the sequence of the nucleic acid of 1); 3) a nucleic acid having a sequence that is at least 95% homologous to the sequence of the nucleic acid of 1) and encoding OTC; 4) an mRNA comprising an OTC open reading frame; 5) an mRNA comprising a codon-optimized OTC open reading frame; 6) a nucleic acid that is partially complementary or fully complementary to any one of 1) - 5); 7) any one of the nucleic acids of 1) - 6) comprises a 5' untranslated region (UTR) sequence and a 3' untranslated region (UTR) sequence; 8) any one of the nucleic acids of 1) - 6) comprises one or more modified nucleotides; 9) any one of the nucleic acids of 1) - 6) is unmodified; 10) any one of the nucleic acids of 1) - 6) comprises a circular RNA or a self-replicating RNA form.

2. The mRNA of claim 1, wherein The modified nucleotides comprise at least one of pseudouridine, 5-methoxyuridine, 5-methylcytidine, 2-thiouridine, N6-methyladenosine (m6A), N1-methyladenosine (mlA), 2'-O-methylation, N6-methyladenosine (m6A), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (5hmC), N4-methylcytidine (m4C), 7-methylguanosine (m7G), N2-methylguanosine (m2G), N1-methylpseudouridine, m2,7G, m2,2,7G, and Nm; Preferably, the pseudouridine comprises at least one of 4-thiopseudouridine, 2-thiopseudouridine, 1-carboxymethylpseudouridine, 1-propynylpseudouridine, 1-taurinomethylpseudouridine, N1-methylpseudouridine, 4-thio-1-methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine, 2-thio-dihydrouridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thiopseudouridine; Preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the nucleotides of the modified nucleotides are replaced; Preferably, the modified nucleotides comprise different nucleotide modifications in the same mRNA molecule; Preferably, the mRNA further comprises a signal peptide coding sequence, a cap structure, and / or a tail structure; Preferably, the mRNA can increase OTC gene expression by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.

3. A DNA molecule, characterized in that, The DNA molecule is transcribed to obtain the mRNA of claim 1 or 2.

4. A recombinant expression vector, characterized in that, containing the mRNA of claim 1 or 2 or the DNA molecule of claim 3; Preferably, the backbone vector of the recombinant expression vector is Puc57, pAAV-MCS, pcDNA 3.1(+), pCMV-MCS, pEGFP-CTSB or pLVX-PAX1. Preferably, the promoter of the recombinant expression vector is Lac lactose operon, TAC promoter, TRC promoter or T7 promoter.

5. A transformant characterized in that, The mRNA of claim 1 or 2, the DNA molecule of claim 3, the recombinant expression vector of claim 4 or the transformant of claim 5.

6. A formulation characterized in that, The mRNA of claim 1 or 2, the DNA molecule of claim 3, the recombinant expression vector of claim 4 or the transformant of claim 5, and a delivery agent; Preferably, the delivery agent comprises a liposome nanoparticle (LNP), a liposome, a polymer, a micelle, a plasmid, a virus or any combination thereof; Preferably, the LNP is selected from a cationic lipid, a helper phospholipid, a sterol lipid, a polyethylene glycol (PEG) modified lipid; Preferably, the cationic lipid is selected from one or more of 4-(N,N-dimethylamino)butanoic acid (dilinoleyl) methyl ester (DLin-MC3-DMA), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine (DLin-KC2-DMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), MC3, DODAC, DDAB, DODMA, Dlin-DAC, C12-200, DODAP, HGT5000, HGT5001, XTC, ALNY-100, cKK-E12; Preferably, the sterol lipid is selected from one or more of cholesterol, cholesterol ester, a sterol hormone, a sterol vitamin and a phytosterol, more preferably one or more of cholesterol, cholesterol ester and phytosterol, most preferably cholesterol; Preferably, the helper phospholipid is selected from one or more of DSPC, DOPC, DPPG, DOPS and DOPE, more preferably DSPC and / or DOPS, most preferably DSPC; Preferably, the polyethylene glycol (PEG) modified lipid is selected from one or more of DAG-PEG, DAA-PEG, DMG-PEG, Cer-PEG and DSPE-PEG, more preferably PEG-DMG; preferably, the relative molecular mass of the PEG is 2000-5000; Preferably, the LNP is selected from MC3, DSPC, cholesterol, mPEG2000-DMG. Preferably, the average particle size of the liposome nanoparticle is 10-500 nm; preferably 80-120 nm; Preferably, the mRNA, the DNA molecule or the recombinant expression vector and the liposome nanoparticle are complexed to form a complex particle; Preferably, at least 95% of the mRNA, the DNA molecule or the recombinant expression vector is encapsulated in the LNP; Preferably, the mRNA, the DNA molecule or the recombinant expression vector is completely encapsulated in the LNP; Preferably, the diameter of the complex particle is 80-120 nm; Preferably, the preparation further comprises a pharmaceutically acceptable excipient; Preferably, the preparation comprises a lyophilized agent, an injection agent; Preferably, the preparation is administered by intravenous, intramuscular, subcutaneous or local route; Preferably, the preparation is an injection agent, and the administration is intravenous injection.

7. A pharmaceutical composition, characterized by, The mRNA of claim 1 or 2, the DNA molecule of claim 3, the recombinant expression vector of claim 4, the transformant of claim 5, the preparation of claim 6, preferably, the pharmaceutical composition further comprises a drug for treating and preventing liver fibrosis and / or a drug for treating HCC; Preferably, the drug for treating liver fibrosis comprises an FXR agonist, a thyroid hormone beta receptor (THR-beta) agonist, an FGF21 / 19 inhibitor, an acetyl coenzyme A carboxylase (ACC) inhibitor, an siRNA, an shRNA, an ASO RNA; Preferably, the drug for treating HCC comprises Sorafenib, Lenvatinib, Apatinib, Regorafenib, PD-1 monoclonal antibody, PD-L1 monoclonal antibody, CTLA4 monoclonal antibody, TIGIT monoclonal antibody, LAG3 monoclonal antibody, VEGF monoclonal antibody, TCR-T cell, CAR-T cell, CAR-macrophage, CAR-NK cell, siRNA, shRNA, ASO RNA.

8. A method of preparing mRNA, characterized in that Transcription of the DNA molecule of claim 3 or the recombinant expression vector of claim 4 to obtain mRNA.

9. A method for increasing the amount of OTC expression in a cell, comprising, Introducing the mRNA of claim 1 or 2, the DNA molecule of claim 3, or the recombinant expression vector of claim 4 into a cell.

10. Use of the mRNA of claim 1 or 2, the DNA molecule of claim 3, the recombinant expression vector of claim 4, or the transformant of claim 5, the preparation of claim 6, the pharmaceutical composition of claim 7 in the manufacture of a medicament for: 1) diagnosing, preventing or treating liver fibrosis; 2) inhibiting or improving the progression of liver fibrosis; 3) diagnosing or treating liver cancer; 4) promoting the differentiation of liver cancer cells into hepatocytes with partial liver function, restoring liver function, and prolonging the survival time of HCC patients; 5) at least one of reducing the content of glutamic-pyruvic transaminase, reducing the content of glutamic-oxaloacetic transaminase, reducing the content of hydroxyproline, reducing the content of alpha-SMA or inhibiting the expression of alpha-SMA, reducing the content of Col1a1, Col1a2 and Acta2 or inhibiting the expression of Col1a1, Col1a2 and Acta2, reducing the content of AFP, reducing the concentration of blood ammonia; 6) increasing the content or expression of Albumin, HNF4a, CYP1A2 and CYP3A4; 7) at least one of increasing the ability of hepatocytes to polymerize glucose molecules, increasing the ability of hepatocytes to transport cholesterol, and increasing the reserve function of hepatocytes.

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