Nucleic acid medicine for treating hyperuricemia-related diseases as well as preparation method and application of nucleic acid medicine

Abstract

The invention relates to an mRNA-liposome compound, the mRNA-liposome compound comprises liposome and nucleic acid, and the nucleic acid comprises at least one mRNA (messenger Ribonucleic Acid) for coding urate oxidase or recombinant urate oxidase; wherein the lipidosome comprises at least two lipids including a core lipid, the lipidosome has pKa of 6.0-6.3, and the molar ratio of the core lipid in the total molar weight of the lipidosome is not less than 15%.

Description

A nucleic acid drug for treating hyperuricemia-related diseases and its preparation method and use Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology. Specifically, the present invention relates to a nucleic acid drug for treating diseases related to hyperuricemia, and a preparation method and use thereof. More specifically, the present invention relates to an mRNA-liposome complex and a preparation method, pharmaceutical composition, use thereof, a method for treating and / or preventing related diseases caused by hyperuricemia, and a method for delivering mRNA primarily to liver cells. Background Art

[0002] Hyperuricemia (HUA) is a chronic metabolic disease caused by impaired purine metabolism. It primarily results from elevated blood uric acid levels due to insufficient renal excretion, excessive secretion, or insufficient intestinal excretion. In the human body, uric acid is the final metabolite of purines. Because uric acid is slightly soluble in water, it easily deposits as urate crystals in various tissues, including joints, cartilage, and kidneys, leading to various diseases such as gout and nephritis.

[0003] Research has found that among mammals, only humans, birds, and some primates use uric acid as the final product of purine metabolism. Most other mammals use urate oxidase (Uox), also known as uricase, to further break down uric acid into water-soluble allantoin for excretion. During human evolution, a nonsense mutation occurred in the Uox gene, rendering it a pseudogene in humans and no longer expressing a functional protein.

[0004] There are three main categories of clinical anti-hyperuricemia drugs. One category is to inhibit uric acid production by inhibiting xanthine oxidase activity, thereby reducing blood uric acid concentration, such as allopurinol and febuxostat. The second category is to reduce blood uric acid concentration by promoting uric acid excretion, such as benzbromarone. However, these small molecule drugs require lifelong medication and have key problems such as drug resistance, and even cause fatal toxic side effects. The third category is urate oxidase drugs, which are protein therapies. They mainly catalyze the slightly water-soluble uric acid into water-soluble allantoin through the injection of urate oxidase in the body. At present, the urate oxidase drugs on the market still have problems such as short half-life and strong immunogenicity.

[0005] Compared to protein therapy, using mRNA to mediate protein expression as a replacement for therapeutic proteins has several potential advantages. mRNA can be expressed directly in the cytoplasm without entering the nucleus, avoiding the risk of genomic integration. On the other hand, by appropriately modifying the regulatory sequence optimization system, the stability and translation efficiency of mRNA can be significantly improved. Efficient delivery systems can enable mRNA to be rapidly taken up and expressed in the cytoplasm. At the same time, as the smallest gene carrier, mRNA has low immunogenicity and will not be degraded by antibody reactions. It can be translated multiple times to improve efficiency. Compared to protein, the production, preparation and purification process of mRNA is simpler, faster, and has lower production costs.

[0006] The efficacy and safety of lipid nanoparticles have been well-documented over decades of nanomedicine research. The first commercially available siRNA drug and the three recent mRNA-based COVID-19 vaccines (mRNA-1273, BNT162b2, and CVnCoV) all used lipid nanoparticles as delivery vehicles.

[0007] Therefore, there is an urgent need to develop a safe, effective and highly efficient mRNA drug for treating hyperuricemia.

[0008] Summary of the Invention

[0009] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, one object of the present invention is to provide a nucleic acid drug that can effectively treat hyperuricemia or other related diseases caused by elevated uric acid.

[0010] In a first aspect, the present invention provides an mRNA-liposome complex, comprising: a liposome and a nucleic acid, wherein the nucleic acid comprises at least one mRNA; wherein the mRNA encodes uricase or recombinant uricase; and the liposome comprises at least two lipids including a core lipid, wherein the molar percentage of the core lipid in the total molar weight of the liposome is not less than 15%.

[0011] In a second aspect of the present invention, the present invention provides a method for preparing the mRNA-liposome complex described in the first aspect, comprising: performing a first mixing treatment on the liposome and the nucleic acid to obtain the mRNA-liposome complex.

[0012] In the third aspect of the present invention, the present invention provides a pharmaceutical composition, which comprises: the mRNA-liposome complex described in the first aspect or the mRNA-liposome complex prepared according to the method described in the second aspect, and optionally a pharmaceutically acceptable excipient or carrier.

[0013] In the fourth aspect of the present invention, the present invention proposes a use of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a drug, wherein the drug has at least one of the following effects: introducing the mRNA into cells, delivering the mRNA mainly to the liver, increasing the expression or activity of related proteins, and preventing and / or treating at least one of related diseases caused by hyperuricemia.

[0014] In the fifth aspect of the present invention, the present invention proposes the use of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect in introducing the mRNA into cells, delivering the mRNA mainly to the liver, increasing the expression or activity of related proteins, and preventing and / or treating related diseases caused by hyperuricemia.

[0015] In the sixth aspect of the present invention, the present invention proposes an mRNA-liposome complex as described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition as described in the third aspect, for introducing the mRNA into cells, delivering the mRNA mainly to the liver, increasing the expression or activity of related proteins, and preventing and / or treating at least one of related diseases caused by hyperuricemia.

[0016] In the seventh aspect of the present invention, the present invention proposes a method for treating and / or preventing related diseases caused by hyperuricemia, which comprises: administering to a subject a pharmaceutically acceptable amount of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0017] In the eighth aspect of the present invention, the present invention proposes a method for delivering mRNA mainly to liver cells, the method comprising: administering to a subject a pharmaceutically acceptable amount of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0018] In a ninth aspect, the present invention provides a method for increasing the expression or activity of uricase or recombinant uricase, the method comprising: administering to a subject a pharmaceutically acceptable amount of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0019] In the tenth aspect of the present invention, the present invention provides a method for treating and / or ameliorating one or more symptoms of diseases related to hyperuricemia in humans, the method comprising administering to the human a therapeutically effective amount of the mRNA-liposome complex described in the first aspect or the pharmaceutical composition described in the second aspect, wherein the mRNA in the mRNA-liposome complex encodes recombinant uricase and / or uricase. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is the structural formula of core lipid A1-D1-5 in Example 2 of the present invention;

[0021] FIG2 is an electron micrograph of lipid nanoparticles mUox@iLAND in Example 3 of the present invention;

[0022] FIG3 shows the stability of lipid nanoparticles mUox@iLAND in Example 3 of the present invention;

[0023] FIG4 is the pKa of the lipid nanoparticles mUox@iLAND in Example 4 of the present invention;

[0024] FIG5 is a flow cytometer analysis of the intracellular effect of lipid nanoparticles in Example 5 of the present invention;

[0025] FIG6 is a confocal laser scanning microscopy image of the intracellular entry of lipid nanoparticles in Example 5 of the present invention;

[0026] FIG7 is a Western blot analysis of the expression of Uox protein in cells of the mUox@iLAND group in Example 6 of the present invention;

[0027] FIG8 is a Western blotting analysis of the expression of Uox proteins in cells of the Uox1@iLAND, Uox2@iLAND, and Uox3@iLAND groups in Example 6 of the present invention;

[0028] FIG9 shows the distribution of mUox@iLAND in animals observed by fluorescence imaging in Example 7 of the present invention;

[0029] FIG10 shows the expression of mUox@iLAND in animals observed by bioluminescent imaging in Example 7 of the present invention;

[0030] FIG11 shows the quantitative results of fluorescence quantification and bioluminescence imaging in Example 7 of the present invention;

[0031] FIG12 shows the enrichment of mRNA in mUox@iLAND in the liver in Example 7 of the present invention (6 hours);

[0032] FIG13 shows the enrichment of mRNA in mUox@iLAND in the liver (24 hours) in Example 7 of the present invention;

[0033] FIG14 shows the changes in uric acid concentration in the serum of each group of animals before and after modeling in Example 8 of the present invention;

[0034] FIG15 shows the changes in serum uric acid concentrations in each group of animals during treatment in Example 8 of the present invention;

[0035] FIG16 shows the changes in body weight of animals in each group during treatment in Example 8 of the present invention;

[0036] FIG17 is an analysis of serum biochemical indicators of animals in each group at the treatment endpoint in Example 8 of the present invention;

[0037] FIG18 shows the changes in uric acid concentration in the serum of each group of animals before and after modeling in step 1 of Example 9 of the present invention;

[0038] FIG19 shows the changes in uric acid concentration in the serum of each group of animals during the treatment process of step 1 in Example 9 of the present invention;

[0039] FIG20 shows the weight changes of animals in each group during the treatment process of step 1 in Example 9 of the present invention;

[0040] FIG21 is an analysis of serum biochemical indicators of animals in each group at the treatment endpoint of step 1 in Example 9 of the present invention;

[0041] FIG22 shows the metabolomics analysis of small molecule metabolism in each group of animals after treatment in step 1 of Example 9 of the present invention;

[0042] FIG23 shows the results of pathological section observation of animal tissues in each group in step 1 of Example 9 of the present invention;

[0043] FIG24 shows the changes in uric acid concentration in the serum of each group of animals during the treatment process of step 2 in Example 9 of the present invention. DETAILED DESCRIPTION

[0044] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0045] It should be noted that, for the structural formula and chemical formula descriptions in the embodiments or embodiments of the present invention, the present invention is intended to cover all replacements, modifications and equivalent technical solutions, which are all within the scope of the present invention as defined in the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described in the present invention can be used to practice the present invention. The present invention is in no way limited to the methods and materials described in the present invention. In the event that one or more of the combined documents, patents and similar materials differ from or contradict the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present invention shall prevail.

[0046] It will be further appreciated that certain features of the invention, which for clarity are described in the context of separate embodiments or implementations, may also be provided in combination in a single embodiment or implementation. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment or implementation, may also be provided separately or in any suitable subcombination.

[0047] Unless otherwise specified, technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the present invention belongs, and unless otherwise specified, all patent publications cited in the entire disclosure of the present invention are incorporated herein by reference in their entirety.

[0048] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0049] As used herein, the compounds of the present invention also include isotopically labeled compounds of the present invention, which are identical to those described herein except for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Exemplary isotopes that may also be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine.

[0050] Compounds of the invention containing the aforementioned isotopes and / or other isotopes of other atoms, as well as pharmaceutically acceptable salts of the compounds, are included within the scope of the present invention. The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers, and mixtures thereof, such as racemic mixtures, are also included within the scope of the present invention.

[0051] Unless otherwise indicated, structures depicted herein are also meant to include all isomers (e.g., enantiomers, diastereomeric atropisomers), and geometric (or conformational) forms of the structure. Therefore, single stereochemical isomers as well as enantiomeric mixtures, diastereomeric mixtures, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention.

[0052] Thus, as described herein, the compounds of the invention may exist in the form of one of the possible isomers, rotamers, atropisomers, tautomers or mixtures thereof, for example, as substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates or mixtures thereof.

[0053] As used herein, the term "solvate" refers to an association formed between one or more solvent molecules and a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed when the solvent molecule is water.

[0054] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0055] As used herein, the term "pharmaceutically acceptable salts" refers to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art.

[0056] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0057] As used herein, the term "pharmaceutically acceptable excipient or carrier" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, salt, drug stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, colorant, or a combination thereof, which are known to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except in the case where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is encompassed.

[0058] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0059] In this article, the terms "related diseases caused by hyperuricemia" and "related diseases caused by elevated uric acid" are synonymous, both referring to diseases caused by elevated uric acid levels, including but not limited to hyperuricemia, gout or other related diseases caused by elevated uric acid (such as gouty nephropathy, gouty vasculopathy, gouty cardiomyopathy, etc.).

[0060] mRNA-liposome complex

[0061] In a first aspect, the present invention provides an mRNA-liposome complex, comprising: a liposome and a nucleic acid; wherein the nucleic acid comprises at least one mRNA encoding urate oxidase (UOX) or recombinant urate oxidase; and the liposome comprises at least two lipids including a core lipid, wherein the molar percentage of the core lipid in the total molar weight of the liposome is not less than 15%.

[0062] According to an embodiment of the present invention, the core lipid is the main skeleton of the mRNA-liposome complex, ensuring that the mRNA-liposome complex can better cross the cell membrane, is neutral in the human peripheral environment (blood, tissue fluid), has low toxicity, and has strong biocompatibility. It can be ionized in the acidic environment (endosomes / lysosomes) within the cell, and can exert an excellent endosomal escape effect, effectively release mRNA, and improve the therapeutic effect of mRNA. Thus, the mRNA-liposome complex can efficiently deliver mRNA to the animal body, and effectively cross the cell membrane, improve the efficiency of mRNA entry into the cell, thereby improving protein expression levels; compared to the mRNA-liposome complex that does not contain the core lipid or the molar amount of the core lipid is less than 15%, the mRNA-liposome complex of the present invention has an increased efficiency of at least 20%. In addition, the mRNA-liposome complex of the present invention has excellent biocompatibility and degradability, and has no obvious toxic side effects, and has good clinical application prospects.

[0063] In some optional embodiments of the present invention, the mRNA-liposome complex may further include at least one of the following technical features:

[0064] In some optional embodiments of the present invention, the liposomes include core lipids, auxiliary lipids, steroids and PEG lipids, and the molar ratio of the core lipids, auxiliary lipids, steroids and PEG lipids is (20-60):(10-50):(30-50):(0.5-2.5).

[0065] In some optional embodiments of the present invention, the liposomes have a pKa of 6.0 to 6.3. This ensures that the mRNA-liposome complex is neutral in the human peripheral environment (blood, tissue fluid), has low toxicity, and is highly biocompatible. Furthermore, the complex can be ionized in the acidic intracellular environment (endosomes / lysosomes), enabling excellent endosomal escape, effectively releasing the mRNA, and enhancing the therapeutic efficacy of the mRNA.

[0066] In some optional embodiments of the present invention, the core lipid is selected from the compound represented by formula (I) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (I);

[0067]

[0068] In some optional embodiments of the present invention, the mRNA encodes a protein that can be expressed in the liver. The inventors have experimentally discovered that, compared to traditional liposome-based drugs, the mRNA-liposome complex of the present invention can efficiently deliver mRNA and other nucleic acid molecules to the liver or primarily target the liver, and can also increase the content or expression of mRNA and other nucleic acid molecules in the liver.

[0069] As used herein, the term "targeting" is non-specific. The term "liver-targeted" refers to mRNA delivery to the liver, where it can express a large amount of the corresponding protein, which primarily exerts its biological activity in the liver. Liver-targeted mRNA is non-specific; the mRNA can also be delivered to other organs of the body to express the corresponding protein, but is primarily delivered to the liver.

[0070] In some optional embodiments of the present invention, the urate oxidase (UOX) or recombinant uricase has an active urate oxidase (UOX) or recombinant uricase that lowers uric acid levels. Thus, the mRNA-liposome complex of the present invention can deliver mRNA to the liver, achieving substantial expression of the recombinant uricase or uricase in the liver, thereby maintaining uric acid levels in patients with hyperuricemia at normal levels. Furthermore, the mRNA-liposome complex of the present invention can maintain efficacy for several weeks, surpassing commercially available recombinant uricase drugs (e.g., rasburicase).

[0071] As used herein, the term "recombinant uricase" refers to a recombinant protein containing a functional fragment of uricase. A "functional fragment" refers to a fragment of uricase that is partially or completely free of uricase and retains its biological activity, i.e., the ability to break down uric acid into allantoin.

[0072] Those skilled in the art will know that recombinant protein refers to a protein obtained by applying recombinant DNA or recombinant RNA technology.

[0073] Illustratively, the urate oxidase is selected from at least one of mouse, primate, bovine, horse, dairy cow, porcine, sheep, goat, dog, cat, rabbit and camel sources, preferably at least one of mouse and primate sources.

[0074] In some optional embodiments of the present invention, the uricase or recombinant uricase comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 6, or an amino acid sequence having at least 90% homology thereto; preferably, the uricase comprises an amino acid sequence having at least 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% homology to any one of SEQ ID NOs: 1 to 6.

[0075] In some optional embodiments of the present invention, the mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 7 to 12, or a nucleotide sequence having at least 90% sequence similarity (similarity or identity) thereto; preferably, the mRNA comprises a nucleotide sequence having at least 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% similarity to SEQ ID NOs: 7 to 12. Alternatively, the mRNA has a similarity with the nucleotide sequence as shown in any one of SEQ ID NOs: 7 to 12 with a difference of 1-15 (preferably 1-10) nucleotides.

[0076] In this article, the nucleotide sequence shown in any one of SEQ ID NOs: 7 to 12 is the coding region (CDS) sequence in the mRNA. It should be noted that "the mRNA comprises the nucleotide sequence shown in any one of SEQ ID NOs: 7 to 12" means that in addition to the nucleotide sequence shown in any one of SEQ ID NOs: 7 to 12, the mRNA may further comprise a non-coding region sequence. The specific non-coding region sequence is not limited. As long as the mRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 7 to 12, it is within the scope of protection of the present invention.

[0077] As used herein, the terms "identity," "homology," "similarity," "identity," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC).

[0078] As used herein, the term "at least 90% sequence homology" or "at least 90% sequence similarity" refers to at least 90%, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% homology or similarity to the respective reference sequence.

[0079] In some optional embodiments of the present invention, the 5' end of the mRNA is connected to a 5'UTR; and / or, the 3' end of the mRNA is connected to a 3'UTR.

[0080] Exemplarily, the 5'UTR has a nucleotide sequence as shown in SEQ ID NO:13.

[0081] Exemplarily, the 3'UTR has a nucleotide sequence as shown in SEQ ID NO:14.

[0082] In some optional embodiments of the present invention, the molar ratio of the core lipid in the total lipid is not less than 15% and not more than 45%; preferably, the molar ratio of the core lipid, the auxiliary lipid, the steroid and the PEG lipid is (20-60): (10-50): (30-50): (0.5-2.5). As a result, the delivery ability of the mRNA molecule to the liver can be further improved, and the mRNA cellular entry efficiency and protein expression level can be further improved. In particular, when the mRNA is an mRNA encoding recombinant uricase or uricase, the mRNA can be mainly delivered to the liver, achieving a large amount of expression of the recombinant uricase or uricase in the liver, and then uric acid can be decomposed into allantoin, effectively treating hyperuricemia. Compared with those mRNA-liposome complexes that do not contain the core lipid, the presence of the core lipid (when the molar ratio in the total lipid is not less than 15%) increases the cellular entry efficiency of the mRNA-liposome complex by at least 20%.

[0083] As used herein, the term "major delivery" means that after systemic administration of the mRNA-liposome complex, most of the mRNA is delivered to liver cells, wherein the majority includes at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%.

[0084] In some optional embodiments of the present invention, the molar ratio of the core lipid, the auxiliary lipid, the steroid and the PEG lipid is (30-39):(9-15):(40-50):(0.5-1).

[0085] Illustratively, the molar ratio of the core lipid, the helper lipid, the steroid, and the PEG lipid is (30-36):(9-11):(40-45):(0.5-0.8).

[0086] In some preferred embodiments of the present invention, the molar ratio of the core lipid, the helper lipid, the steroid and the PEG lipid is a molar percentage, that is, the molar ratio of the core lipid, the helper lipid, the steroid and the PEG lipid is 100%.

[0087] In some optional embodiments of the present invention, the helper lipid can be selected from those suitable species well known in the art. For example, it is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM) and diethyl pyrocarbonate (DEPC).

[0088] In some optional embodiments of the present invention, the steroid is selected from at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol and brassicasterol, preferably cholesterol.

[0089] In some optional embodiments of the present invention, the PEG lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterol glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG - At least one of dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-phosphatidylethanolamine (PEG-PE), PEG-succinic diacylglycerol (PEG-S-DAG), PEG-ceramide (PEG-cer), PEG-dialkoxypropyl carbamate and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0090] In some optional embodiments of the present invention, the PEG lipid includes at least one selected from DMG-PEG 2000, DSPE-PEG 2000, DPPE-PEG 2000 and DMA-PEG 2000.

[0091] In some optional embodiments of the present invention, the mass ratio of the liposome to the mRNA is (1-30):1, preferably (10-30):1, and more preferably (10-20):1.

[0092] Method for preparing mRNA-liposome complex

[0093] In a second aspect, the present invention provides a method for preparing the mRNA-liposome complex described in the first aspect, comprising: mixing the liposomes and the nucleic acid to obtain the mRNA-liposome complex. The method according to an embodiment of the present invention can be used to prepare large quantities of mRNA-liposome complexes, and has the advantages of a simple preparation method, low equipment requirements, and high production efficiency.

[0094] In some optional embodiments of the present invention, the above method may further include at least one of the following technical features:

[0095] In some optional embodiments of the present invention, the liposome is obtained by: using an organic solvent to dissolve the core lipid, the helper lipid, the steroid and the PEG lipid, respectively, to obtain a core lipid solution, a helper lipid solution, a steroid solution and a PEG lipid solution, respectively;

[0096] The core lipid solution, the auxiliary lipid solution, the steroid solution, and the PEG lipid solution are mixed in a first buffer solution to obtain the liposome.

[0097] The organic solvent is an alcoholic organic solvent. The alcoholic organic solvent is selected from C1-C4 alcohol organic solvents, such as C1-C2 alcohol organic solvents. The alcoholic organic solvent is selected from at least one of methanol, ethanol, propanol, and butanol; preferably methanol and ethanol; more preferably ethanol, such as anhydrous ethanol.

[0098] In some optional embodiments of the present invention, the first buffer is selected from at least one of a sodium citrate buffer, an acetate buffer and a sodium bicarbonate buffer.

[0099] In some optional embodiments of the present invention, after the core lipid solution, the auxiliary lipid solution, the steroid solution, and the PEG lipid solution are mixed in a buffer solution, they are further dialyzed.

[0100] In some optional embodiments of the present invention, the dialysis treatment is carried out using a dialysis bag of 50 to 500 kD, for example, 60 kD, 70 kD, 80 kD, 90 kD, 100 kD, 110 kD, 120 kD, 130 kD, 140 kD, 150 kD, 200 kD, 250 kD, 300 kD, 350 kD, 400 kD, or 450 kD.

[0101] In some optional embodiments of the present invention, before mixing the liposomes and the mRNA, the mRNA is dissolved using a second buffer.

[0102] In some optional embodiments of the present invention, the second buffer is selected from at least one of sodium citrate buffer, acetate buffer and sodium bicarbonate buffer.

[0103] In some optional embodiments of the present invention, the second buffer solution may further comprise an ethanol aqueous solution with a volume percentage concentration of 20% to 30%.

[0104] Pharmaceutical composition

[0105] In a third aspect, the present invention provides a pharmaceutical composition comprising: the mRNA-liposome complex described in the first aspect, or the mRNA-liposome complex prepared according to the method described in the second aspect, and optionally, a pharmaceutically acceptable excipient or carrier. As described above, the mRNA-liposome complex has advantages such as low cytotoxicity, good biocompatibility, and strong delivery capability. Thus, a pharmaceutical composition containing the mRNA-liposome complex can deliver mRNA to cells in the body, resulting in substantial expression of related proteins and exerting their efficacy, and is therefore useful for treating diseases caused by hyperuricemia.

[0106] Administration of the pharmaceutical compositions of the present invention can be carried out by any acceptable mode of administration. The pharmaceutical compositions of the present invention can be formulated into solid, semisolid, liquid or gaseous formulations, such as injections, lyophilized powders, and the current methods for preparing these dosage forms are known or will be apparent to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated so as to allow the biologically active ingredients contained therein to be bioavailable after administration of the composition to a patient.

[0107] use

[0108] In the fourth aspect of the present invention, the present invention proposes a use of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a drug, wherein the drug has at least one of the following functions: introducing mRNA into cells, delivering mRNA mainly to the liver, increasing the expression or activity of related proteins, and preventing and / or treating related diseases caused by hyperuricemia.

[0109] In some optional embodiments of the present invention, the related protein is selected from at least one of recombinant uricase and uricase.

[0110] In some optional embodiments of the present invention, the related diseases caused by hyperuricemia are selected from at least one of hyperuricemia, gout, gouty nephropathy, gouty vasculopathy and gouty cardiomyopathy.

[0111] In the fifth aspect of the present invention, the present invention proposes the use of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect in at least one of introducing mRNA into cells, delivering mRNA mainly to the liver, increasing the expression or activity of related proteins, and preventing and / or treating related diseases caused by hyperuricemia.

[0112] In some optional embodiments of the present invention, the related protein is selected from at least one of recombinant uricase and uricase.

[0113] In some optional embodiments of the present invention, the related diseases caused by hyperuricemia are selected from at least one of hyperuricemia, gout, gouty nephropathy, gouty vasculopathy and gouty cardiomyopathy.

[0114] In the sixth aspect of the present invention, the present invention proposes an mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect, which is used to introduce mRNA into cells, deliver mRNA mainly to the liver, increase the expression or activity of related proteins, and prevent and / or treat at least one of related diseases caused by hyperuricemia.

[0115] In some optional embodiments of the present invention, the related protein is selected from at least one of recombinant uricase and uricase.

[0116] In some optional embodiments of the present invention, the related diseases caused by hyperuricemia are selected from at least one of hyperuricemia, gout, gouty nephropathy, gouty vasculopathy and gouty cardiomyopathy.

[0117] method

[0118] In a seventh aspect, the present invention provides a method for treating and / or preventing diseases related to hyperuricemia, comprising administering to a subject a pharmaceutically acceptable amount of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect. As described above, the mRNA-liposome complex has advantages such as low cytotoxicity, good biocompatibility, and strong delivery capability. Thus, a pharmaceutical composition containing the mRNA-liposome complex can deliver mRNA to cells in the body, resulting in substantial expression of related proteins and exerting their efficacy, thereby being used to treat diseases related to hyperuricemia.

[0119] In some optional embodiments of the present invention, the related diseases caused by hyperuricemia are selected from at least one of hyperuricemia, gout, gouty nephropathy, gouty vasculopathy and gouty cardiomyopathy.

[0120] The effective or acceptable amount of the mRNA-liposome complex or pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the bioactive ingredient (e.g., bioavailability, metabolism, half-life, etc.), the severity of the disease to be treated in the subject, the subject's weight, the subject's immune status, the route of administration, and the like. For example, depending on the exigencies of the treatment, several divided doses may be administered daily, or the dose may be proportionally reduced.

[0121] The mRNA-liposome complex or pharmaceutical composition of the present invention can be incorporated into a drug suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms, such as liquid, semisolid, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are injectable solutions. The aforementioned mRNA-liposome complex or pharmaceutical composition can be administered by intravenous infusion or injection, or intramuscular injection, or subcutaneous injection.

[0122] In some optional embodiments of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0123] In the eighth aspect of the present invention, the present invention proposes a method for delivering mRNA mainly to liver cells, the method comprising: administering to a subject a pharmaceutically acceptable amount of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0124] In some optional embodiments of the present invention, the administration route of the method is via a systemic administration route.

[0125] In some optional embodiments of the present invention, the cells are derived from mammals.

[0126] In some optional embodiments of the present invention, the mammal is selected from humans or mice.

[0127] In some optional embodiments of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0128] In a ninth aspect, the present invention provides a method for increasing the expression or activity of uricase or recombinant uricase, the method comprising: administering to a subject a pharmaceutically acceptable amount of the mRNA-liposome complex described in the first aspect, the mRNA-liposome complex prepared according to the method described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0129] In some optional embodiments of the present invention, the administration route of the method is via a systemic administration route.

[0130] In some optional embodiments of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0131] In the tenth aspect of the present invention, the present invention provides a method for treating and / or ameliorating one or more symptoms of diseases related to hyperuricemia in humans, the method comprising administering to the human a therapeutically effective amount of the mRNA-liposome complex described in the first aspect or the pharmaceutical composition described in the second aspect, wherein the mRNA in the mRNA-liposome complex encodes recombinant uricase and / or uricase.

[0132] The amino acid sequences or nucleotide sequences involved in the present invention are shown in the following table:

[0133]

[0134]

[0135]

[0136] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0137] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0138] Example 1: Construction of Uox plasmid and preparation of mUox

[0139] 1. Obtain the mouse Uox mRNA sequence (Gene ID: 22262) from the NCBI database and select the CDS region of the sequence, with the specific nucleotide sequence shown in SEQ ID NO: 7 (the amino acid sequence of the protein it encodes is shown in SEQ ID NO: 1). Subsequently, the 3'UTR (nucleotide sequence shown in SEQ ID NO: 14) and 5'UTR region (nucleotide sequence shown in SEQ ID NO: 13) were added to the optimized CDS region to obtain the optimized mUox sequence. This sequence was inserted into the pcDNA3.1 vector to obtain a plasmid stably expressing the Uox gene. This plasmid was transformed into competent Escherichia coli cells, and the competent cell suspension was streaked onto a prepared LB medium plate and incubated at 37°C for 12-16 hours. Subsequently, a single clone was picked and expanded to obtain a plasmid solution stably expressing the Uox gene, which was aliquoted and stored at -80°C. The plasmid was extracted and linearized and in vitro transcribed to obtain mUox.

[0140] 2. The above method was used to prepare Uox1 mRNA, Uox2 mRNA and Uox3 mRNA, respectively, wherein the nucleotide sequence of the CDS region of Uox1 mRNA is shown in SEQ ID NO: 10, and the amino acid sequence of the protein encoded thereby is shown in SEQ ID NO: 4, the nucleotide sequence of the CDS region of Uox2 mRNA is shown in SEQ ID NO: 11, and the amino acid sequence of the protein encoded thereby is shown in SEQ ID NO: 5, and the nucleotide sequence of the CDS region of Uox3 mRNA is shown in SEQ ID NO: 12, and the amino acid sequence of the protein encoded thereby is shown in SEQ ID NO: 6.

[0141] Example 2: Preparation of lipid nanoparticles

[0142] 1) A1-D1-5 (for its specific chemical formula, see Figure 1 ; it can be prepared using conventional methods in the art, similar to A1-D1-5 in other examples), cholesterol, DOPE, and DMG-PEG 2000 were dissolved in anhydrous ethanol to form a 2 mg / mL organic phase solution. The molar percentage of A1-D1-5 was 35.1%, the molar percentage of DOPE was 11.7%, the molar percentage of cholesterol was 52.6%, and the molar percentage of DMG-PEG 2000 was 0.6%.

[0143] 2) The organic phase solution was rapidly injected into 3 volumes of 50 mM sodium citrate buffer solution with pH = 4.0, while being stirred at high speed to form iLAND.

[0144] 3) The mRNAs obtained in Example 1 (mUox, Uox1 mRNA, Uox2 mRNA, and Uox3 mRNA) were mixed with equal volumes of iLAND (the mass ratio of iLAND to mRNA was 15:1) and incubated at 50° C. for 10 minutes.

[0145] 4) The incubated solution was transferred to a 100 kD dialysis bag and dialyzed in 1×PBS for 2 hours to obtain mUox@iLAND, Uox1@iLAND, Uox2@iLAND, and Uox3@iLAND of the present invention.

[0146] Example 3: Characterization and stability testing of lipid nanoparticles

[0147] 10 μL of mUox@iLAND, Uox1@iLAND, Uox2@iLAND and Uox3@iLAND obtained in Example 2 were respectively added dropwise to the copper mesh and allowed to stand for 10 minutes. The excess liquid on the surface was washed off, stained with 2% uranyl acetate 3 times for 1 minute each time, and washed 3 times with PBS. After the copper mesh was fully dried, the size and morphology of mUox@iLAND were observed using a transmission electron microscope (HT7700). The prepared mUox@iLAND solution was placed in a Malvern particle size analyzer, and its particle size and potential were detected by the principle of dynamic light scattering. The particle size was detected on the 1st, 3rd, 6th, 9th, 12th and 15th day after preparation to monitor its stability. The results of mUox@iLAND, Uox1@iLAND, Uox2@iLAND and Uox3@iLAND were similar, and the results of mUox@iLAND are shown, see Figure 2, Figure 3 and Table 1.

[0148] As shown in the transmission electron microscopy results in Figure 2, mUox@iLAND is spherical with a particle size of approximately 200 nm. As shown in Table 1, the hydrated particle size of mUox@iLAND is consistent with the transmission electron microscopy results, approximately 170 nm. The PDI is less than 0.2, indicating good uniformity. The potential of mUox@iLAND is approximately 0 V, making it suitable for in vivo experimental analysis as a therapeutic drug. As shown in Figure 3, mUox@iLAND remained relatively stable over a two-week period.

[0149] Table 1: Hydrated particle size of mUox@iLAND

[0150]

[0151] Example 4: Determination of pKa of lipid nanoparticles (iLAND)

[0152] First, a series of buffer solutions with pH values ​​ranging from 3 to 10 were prepared (150 mM NaCl, 10 mM boric acid solution, 10 mM phosphoric acid solution, and 10 mM citric acid solution). Blank lipid nanoparticles were prepared (see Example 2 for their preparation method, except that no mRNA was added) and diluted in each solution. 2-(p-Toluidino)-6-naphthalenesulfonic acid (TNS) was dissolved in dimethyl sulfoxide (DMSO) to prepare a TNS solution with a TNS concentration of 300 μM. 2 μL of the TNS solution was mixed with 100 μL of lipid nanoparticles. The fluorescence value was measured under the conditions of excitation light at 325 nm and emission light at 435 nm. Figure 4 shows the curve between fluorescence value and pH value, and the results show that the pKa of the lipid nanoparticle iLAND is 6.02.

[0153] Example 5: Endocytosis of lipid nanoparticles

[0154] 1. Lipid nanoparticles were prepared according to the method of Example 2, except that Cy5 fluorescently labeled mRNA (also known as Cy5 Nucleic acid or nucleic acid molecules) was used this time to obtain fluorescently labeled lipid nanoparticles (also known as Cy5 Nucleic acid@iLAND).

[0155] 2. Hepa1-6 cells (purchased from the Chinese Academy of Sciences Cell Bank) were digested with trypsin and resuspended in DMEM complete medium containing 10% fetal bovine serum to adjust the cell density to about 1×10 5Add 1 mL of cell suspension to each well of a 12-well plate and continue incubating in a 37°C, 5% CO2 incubator for 24 hours to allow complete cell attachment. Add the lipid nanoparticles containing 0.8 μg of Cy5 nucleotides prepared in step 1 to the 12-well plate. Perform flow cytometry / laser confocal microscopy analysis 24 hours after transfection.

[0156] Free Cy5 Nucleic acid was used as a negative control. 0.8 μg of Cy5 Nucleic acid was directly added to the well plate and gently shaken. Flow cytometry / laser confocal microscopy analysis was performed 24 hours after transfection.

[0157] In addition, the commercial transfection reagent Lipofectamine2000 was used as a positive control. Specifically, 2.4 μL Lipofectamine2000 and 0.8 μg Cy5 Nucleic acid were added to 100 μL Opti-MEM low serum medium and incubated. After 5 minutes, the two solutions were mixed and incubated for 15 minutes. The well plate cells to be transfected with Lipofectamine2000 were replaced in advance, the DMEM complete medium was replaced with Opti-MEM, and the incubated Cy5 Nucleic acid@Lipofectamine2000 solution was added to the wells and gently shaken. After 4 hours of transfection, 1 mL of DMEM complete medium was added, and the cells were collected after continuing to culture for 20 hours for flow cytometry / laser confocal microscopy analysis.

[0158] For laser confocal microscopy experiments, cells in all experimental groups were stained for nuclei (Hoechst 33342) and endosomes (Lysotracker Green). The cellular entry and endosomal escape of Cy5 Nucleic acid@iLAND were observed under a laser confocal microscope. The results are shown in Figures 5 and 6.

[0159] Flow cytometry results, as shown in Figure 5, show that lipid nanoparticles Cy5 Nucleic acid@iLAND can mediate good cell entry (G3), essentially equivalent to the commercial transfection reagent Lipofectamine 2000 (G4). However, free Cy5 Nucleic acid cannot effectively enter cells due to the lack of a delivery vehicle (G2).

[0160] The results of laser confocal microscopy analysis are shown in Figure 6. Lipid nanoparticles Cy5 Nucleic acid@iLAND can mediate a good cellular entry effect (G3), while Free Cy5 Nucleic acid cannot enter the cells (G2). The laser confocal microscopy results are consistent with the flow cytometry results.

[0161] Example 6: Expression of lipid nanoparticles at the cellular level

[0162] 1. mUox@iLAND, Uox1@iLAND, Uox2@iLAND, and Uox3@iLAND (all non-fluorescently labeled lipid nanoparticles, for specific preparation methods, see Example 2) were transfected into Hepa1-6 cells, and mUox@Lipofectamine2000 was used as a positive control. 24 hours after transfection, the cells were harvested and lysed. Total cell protein was extracted, and protein was quantified using a BCA protein quantification kit (Congwei Century, CW0014S). The expression of Uox protein in the cells was detected by Western blotting. The results are shown in Figures 7 and 8.

[0163] The results are shown in Figure 7. Both the mUox@Lipofectamine2000 group and the mUox@iLAND group expressed Uox protein, proving that the mUox sequence was successfully constructed and could achieve correct expression of Uox protein.

[0164] As shown in Figure 8, the Uox1@iLAND and Uox1@Lipofectamine2000 groups were able to correctly express the Uox1 protein, the Uox2@iLAND and Uox2@Lipofectamine2000 groups were able to correctly express the Uox2 protein, and the Uox3@iLAND and Uox3@Lipofectamine2000 groups were able to correctly express the Uox3 protein. Therefore, it was shown that the expression sequences (Uox1 mRNA, Uox2 mRNA, and Uox3 mRNA) designed in this example can achieve the correct expression of the novel Uox proteins (Uox1, Uox2, and Uox3).

[0165] Example 7: Distribution and expression of lipid nanoparticles (mRNA@iLAND) at the animal level

[0166] C57BL / 6 mice aged 6-8 weeks were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. The mice were randomly divided into 3 groups and treated with PBS, Cy5 Nucleic acid@Lipofectamine2000 prepared in Example 5, and Cy5 Nucleic acid@iLAND prepared in Example 5 through the tail vein. Fluorescence imaging and bioluminescence imaging were performed on the animals 3 hours, 6 hours, and 24 hours after injection to monitor the distribution and expression of mRNA. The mice were killed and dissected at different time points, and the submandibular gland, thymus, heart, liver, spleen, lung, and kidney were taken for imaging to more clearly observe the distribution and expression of mRNA in various organs. The liver tissue dissected out at 6 hours and 24 hours was embedded in OCT gel, and the embedded tissue was then frozen and sectioned. The sections were stained with DAPI and FITC-labeled phalloidin, and then fluorescence observation was performed using a laser confocal microscope. The results are shown in Figures 9 to 13.

[0167] The in vivo drug distribution and expression results are shown in Figures 9 and 10, respectively. The results show that through intravenous administration, Cy5 Nucleic acid@Lipofectamine2000 (G2) is metabolized extremely rapidly in vivo, lasting for 3-6 hours (Figure 9), and is unable to achieve protein expression function in vivo (Figure 10); while Cy5 Nucleic acid@iLAND (G3) can achieve long-term drug circulation in vivo and can achieve efficient drug expression in the liver.

[0168] The quantitative results analysis in Figure 11 showed that the Cy5 Nucleic acid@iLAND group exhibited long-lasting pharmacokinetic distribution ability and protein expression level in the liver.

[0169] The frozen section results in Figures 12 and 13 show that the fluorescently labeled nucleic acid molecules can be effectively enriched in the liver tissue, reaching a peak at 6 hours and being able to maintain for about 24 hours.

[0170] Example 8: Therapeutic Effects of Lipid Nanoparticles (mUox@iLAND) in Model Animals

[0171] Female C57BL / 6 mice, aged 6-8 weeks, were purchased from Spefox (Beijing) Biotechnology Co., Ltd. The mice were randomly divided into four groups and treated differently: one group was maintained normally, while the remaining three groups were induced to develop hyperuricemia. The model mice were subcutaneously injected with a uric acid inhibitor on day -14. On day 0, orbital bleeding was collected from all mice, and serum was isolated for uric acid analysis. After confirming the successful establishment of the model mice, drug administration began: the mice were not established and raised normally, and the administration method was 200 μL PBS injected into the tail vein (Control group, G1); the mice were established, and 14 days after the establishment of the model, the administration method was 200 μL PBS injected into the tail vein (PBS group, G2); the mice were established, and 14 days after the establishment of the model, a certain amount of Allopurinol (Allopurinol is a clinical first-line drug for anti-hyperuricemia, serving as the experimental positive control group) was added to the drinking water when the drug was started, and the mice were allowed to drink freely every day (Allopurinol group, G3); the mice were established, and 14 days after the establishment of the model, the administration method was 200 μL mUox@iLAND prepared in Example 2 injected into the tail vein (mUox@iLAND group, G4), and the administration was only done once. The drugs were administered on the first day. Except for the Allopurinol group, which was administered daily, the other three groups were administered once throughout the entire process. At different time points after administration, blood was collected from all mice's orbits, and the serum was separated and analyzed for uric acid and other biochemical indicators. The weight changes of the mice were recorded throughout the modeling and drug treatment. The results are shown in Figures 14 to 17.

[0172] The results of the model construction are shown in Figure 14. After 14 days of modeling, the uric acid level in the model mice was significantly increased (from 100 μM to 400 μM), indicating that the hyperuricemia mouse model was successfully constructed.

[0173] The results of anti-hyperuricemia treatment are shown in Figure 15. Compared to the PBS group, the Allopurinol and mUox@iLAND groups were able to maintain uric acid levels in mice at a certain level. However, the Allopurinol group required daily administration, while the mUox@iLAND group only required a single dose. These results demonstrate that the lipid nanoparticle mUox@iLAND is highly effective in treating hyperuricemia. Compared to the first-line clinical drug Allopurinol, which requires daily administration, mUox@iLAND requires only a single dose to maintain normal blood uric acid levels for an extended period, demonstrating a significant advantage.

[0174] The weight changes of mice in each group are shown in Figure 16. The weight of mice in the mUox@iLAND group was similar to that in the Control group, indicating that mUox@iLAND has good safety.

[0175] Serum biochemical indicators are shown in Figure 17. mUox@iLAND showed no obvious toxic side effects, indicating that the safety of mUox@iLAND is better than that of the clinical first-line drug Allopurinol.

[0176] Example 9: Therapeutic Effects of Lipid Nanoparticles in Model Animals

[0177] This embodiment differs from embodiment 7 in that a different method is used to construct a hyperuricemia model.

[0178] 1. Therapeutic effects of mUox@iLAND in model animals

[0179] Female C57BL / 6 mice aged 6-8 weeks were purchased from Spefox (Beijing) Biotechnology Co., Ltd. The mice were randomly divided into four groups and treated differently: one group was maintained normally, and the remaining three groups were subjected to hyperuricemia modeling. For the modeling mice, a 0.15% high-purine diet was maintained from day -14, establishing a hyperuricemia model through a continuous high-purine diet. On day 0, orbital bleeding was performed on all mice, and serum was isolated for uric acid testing. After confirming the successful establishment of the model mice, drug administration began: the mice were not established and raised normally, and the administration method was 200 μL PBS injected into the tail vein (Control group, G1); the mice were established, and 14 days after the establishment of the model, the administration method was 200 μL PBS injected into the tail vein (PBS group, G2); the mice were established, and 14 days after the establishment of the model, a certain amount of Allopurinol (Allopurinol is a clinical first-line drug for anti-hyperuricemia, serving as the experimental positive control group) was added to the drinking water when the drug was started, and the mice were allowed to drink freely every day (Allopurinol group, G3); the mice were established, and 14 days after the establishment of the model, the administration method was 200 μL mUox@iLAND prepared in Example 2 injected into the tail vein (mUox@iLAND group, G4), and the administration was only done once. The drugs were administered on the first day. Except for the Allopurinol group, which was administered daily, the other three groups were administered once throughout the entire process. All mice underwent orbital bleeding at various time points after dosing, and serum was isolated and analyzed for uric acid and other biochemical markers. Body weight changes were recorded throughout the modeling and treatment. At the end of the experiment, eyeballs were removed for blood collection, and mice were sacrificed. Liver tissue was obtained for metabolomics analysis, and tissues and organs such as the heart, liver, spleen, lung, and kidney were fixed and analyzed for pathological changes using H&E staining. The results are shown in Figure 18.

[0180] The results of the model construction are shown in Figure 18. After 14 days of modeling, the uric acid level in the model mice was significantly increased (from 100 μM to 400 μM), indicating that the hyperuricemia mouse model was successfully constructed.

[0181] The results of anti-hyperuricemia treatment are shown in Figure 19. Compared to the PBS group, the clinically used first-line drug Allopurinol reduced blood uric acid levels below normal, even to around 0 μM. This suggests that Allopurinol carries certain risks and is not conducive to maintaining normal blood uric acid levels in the human body. However, the mUox@iLAND group, which only required a single dose, was able to maintain uric acid levels in mice at a certain normal level, demonstrating that the lipid nanoparticle mUox@iLAND can achieve safe and effective anti-hyperuricemia treatment, showing a significant advantage.

[0182] The weight changes of mice in each group are shown in Figure 20. The weight of mice in the mUox@iLAND group was similar to that in the Control group, indicating that mUox@iLAND has good safety.

[0183] The serum biochemical results are shown in Figure 21 , and mUox@iLAND showed no obvious toxic side effects.

[0184] The metabolomics analysis results are shown in Figure 22. The metabolomics results indicate that mUox@iLAND mediates excellent anti-hyperuricemia effects, and the molecular metabolic levels in the treated mice are closest to those in the healthy mice control group.

[0185] The pathological section results are shown in Figure 23, indicating that mUox@iLAND has good safety.

[0186] In summary, mUox@iLAND achieves long-term stabilization of blood uric acid levels with a single administration, and its safety and efficacy are superior to those of the first-line clinical drug Allopurinol. mUox@iLAND opens up a new avenue for clinical drug research and development.

[0187] 2. Therapeutic effects of Uox1@iLAND in model animals

[0188] The same method as in step 1 of this example was used to establish a hyperuricemia mouse model, and the therapeutic efficacy of the novel lipid nanoparticle Uox1@iLAND in this mouse hyperuricemia model was evaluated. Mice were randomly divided into four groups and treated differently: Group G1 mice were not modeled and housed normally, with the drug administered via tail vein injection of 200 μL of PBS (Control group); Group G2 mice were administered 200 μL of PBS via tail vein injection 14 days after modeling (PBS group); Group G3 mice were administered daily 14 days after modeling, with a fixed amount of Allopurinol added to their drinking water (Allopurinol group, a first-line clinical anti-hyperuricemia drug, served as the positive control group for the experiment), with free access to water; Group G4 mice were administered 200 μL of Uox1@iLAND prepared in Example 2 via tail vein injection 14 days after modeling (Uox1@iLAND group), with only one dose. At different time points after administration, all mice were bled retro-orbitally, and the serum was separated and the uric acid level was determined.

[0189] The experimental results are shown in Figure 24. After 14 days of modeling, the blood uric acid level of the model mice (G2 group) was significantly improved compared with the unmodeled animals (G1 group), indicating that the hyperuricemia mouse model was successfully constructed; the results of the positive control group (G3) showed that the clinical first-line drug Allopurinol can reduce the blood uric acid level to the same or lower than the normal level; and the Uox1@iLAND group in group G4, which only needed one dose of the drug, was able to maintain the uric acid level in the mice at a normal level, indicating that the lipid nanoparticles Uox1@iLAND can achieve safe and efficient anti-hyperuricemia treatment.

[0190] In summary, Uox1@iLAND can achieve long-term stable blood uric acid levels with a single administration, indicating that the novel Uox protein and novel lipid nanoparticles designed in this invention can be used to regulate uric acid levels, opening up a new avenue for the development of clinical drugs.

[0191] Example 10:

[0192] Female C57BL / 6 mice aged 6-8 weeks were purchased from Spefox (Beijing) Biotechnology Co., Ltd. The mice were randomly divided into four groups and treated differently: one group was maintained normally, and the remaining three groups were subjected to hyperuricemia modeling. For the modeling mice, a 0.15% high-purine diet was maintained from day -14, establishing a hyperuricemia model through a continuous high-purine diet. On day 0, orbital bleeding was performed on all mice, and serum was isolated for uric acid testing. After confirming successful modeling, the mice were treated with medication: the mice were not modeled and housed normally, with 200 μL of PBS injected into the tail vein (Control group, G1); the mice were modeled, and 14 days after modeling, they were administered with 200 μL of PBS injected into the tail vein (PBS group, G2); the mice were modeled, and 14 days after modeling, they were administered with 200 μL of mUox prepared in Example 1 into the tail vein (Free mUox group, G3), and 14 days after modeling, they were administered with 200 μL of mUox@iLAND prepared in Example 2 into the tail vein (mUox@iLAND group, G4). Dosing was performed on day 1, and serum uric acid levels were measured by orbital blood sampling on days 2 (24 hours after dosing), 3 (48 hours after dosing), and 7 (144 hours after dosing). The results of model construction and anti-uric acid treatment are shown in Table 2. After 14 days of modeling, uric acid levels in the model mice were significantly increased (from 100 μM to 400 μM), indicating that the hyperuricemia mouse model was successfully established. Anti-hyperuricemia treatment results showed that compared with the PBS group, the mUox@iLAND group was able to significantly reduce uric acid levels in mice, while the Free mUox group showed no significant change in uric acid levels before and after administration. This indicates that mUox mRNA alone, without the novel lipid nanoparticles as a delivery vehicle, cannot achieve targeted delivery and protein expression in vivo.

[0193] Table 2

[0194]

[0195] Example 11:

[0196] 1) Dissolve A1-D1-5, cholesterol, DOPE, and DMG-PEG 2000 in anhydrous ethanol to form a 2 mg / mL organic phase solution.

[0197] 2) An organic phase was prepared at a molar ratio of 30:10:45:0.5 among A1-D1-5, DOPE, cholesterol, and DMG-PEG 2000 to obtain organic phase 1. An organic phase was prepared at a molar ratio of 70:10:45:0.5 among A1-D1-5, DOPE, cholesterol, and DMG-PEG 2000 to obtain organic phase 2. An organic phase was prepared at a molar ratio of 30:60:45:0.5 among A1-D1-5, DOPE, cholesterol, and DMG-PEG 2000 to obtain organic phase 3. An organic phase was prepared at a molar ratio of 30:10:80:0.5 among A1-D1-5, DOPE, cholesterol, and DMG-PEG 2000 to obtain organic phase 4. The organic phase was prepared according to the molar ratio of A1-D1-5, DOPE, cholesterol, and DMG-PEG 2000 of 30:10:45:5 to obtain organic phase 5.

[0198] 3) The solutions of the organic phases 1-5 were respectively injected into 3 volumes of 50 mM sodium citrate buffer solution with pH=4.0 at high speed while stirring at high speed to form iLAND1, iLAND2, iLAND3, iLAND4, and iLAND5.

[0199] 4) The mRNA (mUox) obtained in Example 1 was mixed with equal volumes of the five iLANDs (the mass ratio of iLAND to mRNA was 15:1) and incubated at 50° C. for 10 minutes.

[0200] 5) The incubated solution was transferred to a 100 kD dialysis bag and dialyzed in 1× PBS for 2 h to obtain mUox@iLAND1, mUox@iLAND2, mUox@iLAND3, mUox@iLAND4, and mUox@iLAND5.

[0201] 6) Five types of mUox@iLAND were transfected into Hepa1-6 cells, respectively. Total cell protein was extracted 24 hours after transfection, and the expression level of mUox was detected by Western blotting.

[0202] The experimental results are shown in Table 3. mUox@iLAND1 was able to achieve efficient protein expression, and its expression level was calculated as 100%. The other four liposomes were unable to achieve efficient protein expression, indicating that the specific lipid composition ratio in the present invention is the key to achieving efficient mRNA expression.

[0203] Table 3

[0204]

[0205]

[0206] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An mRNA-liposome complex, characterized in that: include: Liposomes and nucleic acids; Wherein, the nucleic acid comprises at least one mRNA encoding uricase or recombinant uricase; The liposome comprises at least two lipids including a core lipid, and the molar percentage of the core lipid in the total molar amount of the liposome is not less than 15%.

2. The mRNA-liposome complex according to claim 1, characterized in that The liposome comprises core lipid, auxiliary lipid, steroid and PEG lipid, and the molar ratio of the core lipid, the auxiliary lipid, the steroid and the PEG lipid is (20-60):(10-50):(30-50):(0.5-2.5).

3. The mRNA-liposome complex according to claim 1 or 2, characterized in that The liposome has a pKa of 6.0 to 6.3; Optionally, the core lipid is selected from a compound having a structure as shown in formula (I) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound as shown in formula (I); 4. The mRNA-liposome complex according to any one of claims 1 to 3, characterized in that The uricase or recombinant uricase is mainly expressed in the liver; Optionally, the uricase or recombinant uricase has activity in reducing uric acid levels; Optionally, the recombinant uricase or uricase comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 6, or an amino acid sequence having at least 90% sequence homology thereto.

5. The mRNA-liposome complex according to claim 1 or 2, characterized in that: The mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 7 to 12, or a nucleotide sequence having at least 90% sequence similarity thereto.

6. The mRNA-liposome complex according to any one of claims 1 to 5, characterized in that: The 5' end of the mRNA is connected to the 5'UTR; and / or the 3' end of the mRNA is connected to the 3'UTR.

7. The mRNA-liposome complex according to any one of claims 1 to 6, characterized in that: The molar ratio of the core lipid, auxiliary lipid, steroid and PEG lipid is (20-60):(10-50):(30-50):(0.5-2.5); Preferably, the molar ratio of the core lipid, auxiliary lipid, steroid and PEG lipid is (30-39):(9-15):(40-50):(0.5-1).

8. The mRNA-liposome complex according to claim 2, characterized in that The auxiliary lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE ), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM) and diethyl pyrocarbonate (DEPC); Optionally, the steroid is selected from at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol and brassicasterol, preferably cholesterol; Optionally, the PEG lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, P- At least one of EG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-phosphatidylethanolamine (PEG-PE), PEG-succinic diacylglycerol (PEG-S-DAG), PEG-ceramide (PEG-cer), PEG-dialkoxypropylcarbamate and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA); Optionally, the PEG lipid is selected from at least one of DMG-PEG 2000, DSPE-PEG 2000, DPPE-PEG 2000 and DMA-PEG 2000.

9. The mRNA-liposome complex according to any one of claims 1 to 8, characterized in that The weight ratio of the liposome to the mRNA is (1-30):1, preferably (10-20):

1.

10. A method for preparing the mRNA-liposome complex according to any one of claims 1 to 9, characterized in that: include: The liposome and the nucleic acid are mixed to obtain the mRNA-liposome complex.

11. The method according to claim 10, characterized in that The liposomes are obtained in the following manner: Using an organic solvent to dissolve the core lipid, the auxiliary lipid, the steroid and the PEG lipid, respectively, and obtain a core lipid solution, an auxiliary lipid solution, a steroid solution and a PEG lipid solution, respectively; The core lipid solution, the auxiliary lipid solution, the steroid solution, and the PEG lipid solution are mixed in a first buffer to obtain the liposome; Optionally, the organic solvent is an alcohol organic solvent; Optionally, the alcohol organic solvent is selected from C1 to C4 alcohol organic solvents; Optionally, the alcohol organic solvent is selected from at least one of methanol, ethanol, propanol and butanol, preferably selected from methanol and ethanol, and more preferably ethanol; Optionally, the first buffer is selected from at least one of a sodium citrate buffer, an acetate buffer and a sodium bicarbonate buffer.

12. The method according to claim 10, characterized in that Before mixing the liposomes and the nucleic acid, dissolving the mRNA using a second buffer; Optionally, the second buffer is selected from at least one of a sodium citrate buffer, an acetate buffer and a sodium bicarbonate buffer; Optionally, the second buffer further comprises an ethanol aqueous solution with a volume percentage concentration of 20% to 30%.

13. A pharmaceutical composition, characterized in that include: The mRNA-liposome complex according to any one of claims 1 to 9 or the mRNA-liposome complex prepared according to the method according to any one of claims 10 to 12, and optionally a pharmaceutically acceptable excipient or carrier.

14. Use of the mRNA-liposome complex according to any one of claims 1 to 9, the mRNA-liposome complex prepared according to the method according to any one of claims 10 to 12, or the pharmaceutical composition according to claim 13 in preparing a drug, wherein the drug has at least one of the following effects: Introducing the mRNA into cells, delivering the mRNA mainly to the liver, increasing the expression or activity of related proteins, and preventing and / or treating related diseases caused by hyperuricemia; Optionally, the related protein is selected from at least one of recombinant uricase and uricase; Optionally, the related diseases caused by hyperuricemia are selected from at least one of hyperuricemia, gout, gouty nephropathy, gouty vasculopathy and gouty cardiomyopathy.

15. A method for treating and / or preventing diseases related to hyperuricemia, characterized in that: include: Administering a pharmaceutically acceptable amount of the mRNA-liposome complex according to any one of claims 1 to 9, the mRNA-liposome complex prepared according to the method according to any one of claims 10 to 12, or the pharmaceutical composition according to claim 13 to a subject; Optionally, the related diseases caused by hyperuricemia are selected from at least one of hyperuricemia, gout, gouty nephropathy, gouty vasculopathy and gouty cardiomyopathy; Optionally, the administration route of the method is subcutaneous injection or intravenous injection.

16. A method for delivering mRNA primarily to liver cells, characterized in that: include: Administering a pharmaceutically acceptable amount of the mRNA-liposome complex according to any one of claims 1 to 9, the mRNA-liposome complex prepared according to the method according to any one of claims 10 to 12, or the pharmaceutical composition according to claim 13 to a subject; Optionally, the administration route of the method is via a systemic administration route, preferably subcutaneous injection or intravenous injection; Optionally, the liver cells are derived from mammals; Preferably, the mammal is selected from humans and mice.

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