Polynucleotide encoding uricase, and related composition and method thereof

Abstract

Provided are a nucleic acid molecule encoding uricase, or a functional fragment or variant thereof (comprising a uricase fusion protein). Further provided is a composition containing the nucleic acid molecule, comprising a lipid nanoparticle (LNP), and a related treatment method and use for treating or preventing gout, gouty arthritis, hyperuricemia or tumor lysis syndrome in a subject.

Description

Polynucleotides encoding uricase and related compositions and methods

[0001] This application claims priority to Chinese patent application No. CN202310937175.4 filed on July 27, 2023 and Chinese patent application No. CN202410233818.1 filed on March 1, 2024, which are hereby incorporated by reference in their entirety. Technical Field

[0002] The present disclosure generally relates to nucleic acid molecules encoding urate oxidase, functional fragments or variants thereof. The present disclosure also relates to compositions comprising the nucleic acid molecules, including lipid nanoparticles (LNPs), and related treatment methods and uses for treating or preventing gout, gouty arthritis, hyperuricemia, or tumor lysis syndrome in a subject. Background Art

[0003] Uric acid is a key product in the purine metabolic pathway. The pathway from purine degradation to uric acid production is highly conserved across the biological world. Uricase, an enzyme involved in purine metabolism in most mammals, converts the less soluble uric acid into the more soluble and excreted allantoin. However, the urate oxidase gene has been inactivated during evolution in most higher primates, including humans. Consequently, higher primates like humans have relatively high uric acid levels and are prone to hyperuricemia.

[0004] In recent decades, urate oxidase from other species has proven effective in treating a variety of diseases, the most common of which is gout. Gout is an acute arthritis caused by excessive uric acid levels in the blood, which leads to the deposition of urate crystals in the joints. It is more common in men. According to statistics, the prevalence of gout in Chinese adults is 1.1%. Economic development has led to changes in people's diets and lifestyles, with an increased intake of high-protein and high-purine foods, which in turn increases uric acid production in the liver. Furthermore, dysfunction of urate transporters can reduce uric acid excretion through the kidneys or intestines. These two factors together contribute to the annual increase in the number of gout patients. Urate oxidase can rapidly lower blood uric acid levels in gout patients and help dissolve urate crystals deposited in their joints, reducing pain and providing relief for gout patients.

[0005] In addition, urate oxidase is also widely used in the treatment and prevention of tumor lysis syndrome (TLS). TLS refers to a tumor emergency that occurs spontaneously or during tumor treatment, caused by the rapid lysis of a large number of tumor cells, releasing large amounts of potassium, phosphate, and nucleic acids into the systemic circulation. Common clinical manifestations are hyperuricemia, hyperphosphatemia, hyperkalemia, and hypocalcemia, which can lead to life-threatening complications such as arrhythmias, epilepsy, and renal failure, seriously threatening the life and health of cancer patients. TLS is common in hematological tumors such as acute leukemia (ALL and AML), non-Hodgkin's lymphoma (NHL), etc., and occasionally occurs in solid tumors such as small cell carcinoma, breast cancer, and neuroblastoma.

[0006] Currently, there are two clinically approved uricase drugs. Although both have achieved good efficacy, their clinical application is still limited by side effects caused by immunogenicity. Krystexxa (Pegloticase), a pegylated pig-baboon chimeric uricase, is approved for the treatment of refractory gout refractory to conventional urate-lowering therapy via intravenous injection of 8 mg every two weeks. It has good efficacy in dissolving tophi. However, Krystexxa has received a black box warning from the FDA due to allergic reactions and infusion reactions. In addition, 92% of patients have anti-drug antibodies against Krystexxa in their blood. Elite (Rasburicase), a uricase derived from Aspergillus flavus, is approved for the treatment of hyperuricemia caused by TLS. Due to its short half-life, it requires daily intravenous infusion for five consecutive days. Elite also carries a black box warning for allergic reactions and hemolysis. In addition to high immunogenicity, the high cost of uricase oxidase drugs also limits their clinical application. A single injection of Krystexxa (8mg) costs $25,585.46, while a full course of Elite (5-day treatment) costs $13,624.6. Therefore, the development of safer, more convenient, more accessible, and less immunogenic uricase drugs could provide greater benefits to patients.

[0007] In recent years, with the maturation of in vitro transcription (IVT)-based delivery technologies, as well as liposomes and nanoparticles, mRNA therapy has demonstrated tremendous potential. mRNA prepared in vitro is encapsulated in a vector and delivered to tissues in vivo, where it is internalized by cells. After uptake, the mRNA is recognized by ribosomes and synthesized into the corresponding protein based on its encoded information. Compared to DNA therapies, mRNA therapeutics do not require entry into the cell nucleus, thus eliminating the risk of insertional mutagenesis associated with genomic integration. Compared to protein therapeutics, mRNA therapeutics utilize the cell's native translational machinery, enabling the expression of both secreted and intracellular proteins. Furthermore, the use of modified nucleotides has made mRNA immunogenicity more controllable, significantly improved in vivo stability, more predictable pharmacokinetic properties, and the ability to be produced using more efficient and scalable manufacturing processes. Therefore, for enzyme replacement therapy, mRNA therapeutics can effectively offset the limitations and drawbacks of exogenous protein therapeutics. Using mRNA therapeutics to express urate oxidase appears to offer a promising approach for the treatment of gout and TLS, potentially improving efficacy, safety, and convenience.

[0008] The expression of uricase in mammals (such as rats, mice, hamsters, dogs, cats and cattle) that still retain uricase is limited to the liver and localized in peroxisomes. The recombinant uricase currently on the market is distributed in the periphery after administration and cannot enter the liver cells to clear uric acid. It can only clear uric acid that has been released into the circulation. In order to achieve an effective blood drug concentration, the drug injection dose is relatively high, infusion reactions are prone to occur, anti-drug antibodies are easily produced, and there is a lot of room for improvement in safety. WO2022267086 describes mRNA encoding two uricase listed drugs, Pegloticase and Rasburicase, for the treatment of hyperuricemia and gout, but the natural mRNA used in the invention is recognized to have high immunogenicity in vivo, is easily degraded, and has low bioavailability. At the same time, the mRNA sequence of the invention has not been optimized and is not liver-targeted for delivery. Therefore, the drug potential of the uricase mRNA of the invention is still insufficient.

[0009] In view of this, there is currently no safe and effective mRNA drug that can achieve liver-targeted delivery for the treatment of gout and TLS.

[0010] Summary of the Invention

[0011] The inventors of this application have solved the above-mentioned needs through creative work.

[0012] Specifically, the present disclosure relates to the following technical solutions:

[0013] In one embodiment, the present disclosure relates to a nucleic acid comprising an open reading frame (ORF) encoding uricase, a functional fragment or variant thereof, including a uricase fusion protein.

[0014] In further embodiments, the functional fragment or variant of uricase is a truncated uricase or a uricase fusion protein that retains uricase activity.

[0015] In yet further embodiments, the uricase has the amino acid sequence of any one of SEQ ID NOs: 1-21, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to any one of SEQ ID NOs: 1-21.

[0016] In yet further embodiments, the uricase fusion protein is a fusion protein of a signal peptide and an optional spacer sequence with uricase, or and / or a fusion protein of a peroxisome localization peptide with uricase.

[0017] In yet a further embodiment, the uricase fusion protein is a fusion protein comprising a signal peptide, a spacer sequence, and uricase from the N-terminus to the C-terminus.

[0018] In yet further embodiments, the uricase fusion protein is a fusion protein comprising, from N-terminus to C-terminus, a signal peptide, a spacer, a half-life extending element, an optional linker, and uricase; a fusion protein comprising, from N-terminus to C-terminus, a signal peptide, a spacer, a uricase, an optional linker, and a half-life extending element; a fusion protein comprising, from N-terminus to C-terminus, a signal peptide, a half-life extending element, an optional linker, and uricase; or a fusion protein comprising, from N-terminus to C-terminus, a signal peptide, a spacer, a uricase, an optional linker, and a half-life extending element. A fusion protein comprising a long element, optionally wherein the uricase fusion protein is a fusion protein comprising a signal peptide, a spacer sequence, a half-life extending element, a linker and uricase from the N-terminus to the C-terminus, a fusion protein comprising a signal peptide, a spacer sequence, a uricase oxidase, a linker, a half-life extending element from the N-terminus to the C-terminus, a fusion protein comprising a signal peptide, a half-life extending element, a linker and uricase from the N-terminus to the C-terminus, or a fusion protein comprising a signal peptide, uricase, a linker, a half-life extending element from the N-terminus to the C-terminus.

[0019] In yet further embodiments, the nucleic acid is DNA or mRNA.

[0020] In another embodiment, the present disclosure relates to a vector or plasmid comprising the nucleic acid of any of the preceding embodiments.

[0021] In yet another embodiment, the present disclosure relates to a host cell comprising the nucleic acid or vector or plasmid of any one of the preceding embodiments.

[0022] In yet another embodiment, the present disclosure relates to a pharmaceutical composition comprising the nucleic acid of any of the preceding embodiments and a delivery agent.

[0023] In yet another embodiment, the present disclosure relates to a method for reducing uric acid levels in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the nucleic acid or pharmaceutical composition according to any one of the preceding embodiments.

[0024] In yet another embodiment, the present disclosure relates to a method for treating or preventing gout, gouty arthritis, hyperuricemia or tumor lysis syndrome in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the nucleic acid or pharmaceutical composition according to any one of the preceding embodiments.

[0025] In yet another embodiment, the present disclosure relates to a method for enhancing expression of an open reading frame (ORF) encoding a uricase fusion protein in a nucleic acid, wherein the uricase fusion protein is a fusion protein of a signal peptide and uricase, wherein the signal peptide is fused to the N-terminus of the uricase, the method comprising inserting a coding sequence for a spacer sequence between the coding sequence for the signal peptide and the coding sequence for the uricase in the ORF.

[0026] In yet another embodiment, the present disclosure relates to a method for enhancing expression of an open reading frame (ORF) encoding a uricase fusion protein in a nucleic acid, wherein the uricase fusion protein comprises, from the N-terminus to the C-terminus, a signal peptide, an optional spacer sequence, a half-life extending element, an optional linker, and a uricase, or comprises, from the N-terminus to the C-terminus, a signal peptide, an optional spacer sequence, a uricase, an optional linker, and a half-life extending element, the method comprising adding a coding sequence for a half-life extending element and a coding sequence for an optional linker to the 5' or 3' end of the coding sequence for the uricase in the ORF.

[0027] The details of the present application are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are now described. Other features, objects and advantages of the present application will be apparent from the specification and claims. In the specification and the appended claims, the singular also includes the plural, unless the context clearly provides otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present application belongs. All patents and publications cited in this specification are incorporated herein by reference in their entirety.

[0028] The contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic diagram of an exemplary mRNA structure

[0030] Figure 2 is an exemplary plasmid map.

[0031] Figure 3 shows changes in serum uric acid levels in rats with hyperuricemia induced by a high-uric acid diet / potassium oxonate after intravenous injection of a uricase mRNA composition. The uricase mRNA composition in the figure is uricase mRNA encapsulated in LNP formulation 1. The dosages indicated in the figure are the mRNA dosages in the composition.

[0032] Figures 4a-b show serum uricase activity in normal mice after intravenous injection of a uricase mRNA composition. Figures 4c-d show serum uric acid levels in normal mice after intravenous injection of a uricase mRNA composition. The uricase mRNA composition in the figures is uricase mRNA encapsulated in LNP formulation 1. The dosages indicated in the figures are the mRNA dosages in the composition.

[0033] Figure 5 shows the serum uric acid levels in normal mice after administration of uricase mRNA compositions encapsulated in different LNP formulations. The dosages shown in the figure are the dosages of the mRNA in the compositions.

[0034] [Corrected 26.08.2024 in accordance with Rule 91] Figure 6 shows serum uric acid levels in normal mice after administration of a uricase mRNA composition via different routes. The uricase mRNA composition described in the figure is uricase mRNA encapsulated in LNP formulation 2. The dosages indicated in the figure are the mRNA dosages in the composition.

[0035] [Corrected 26.08.2024 in accordance with Rule 91] Figure 7a shows the changes in serum uric acid levels in rats with hyperuricemia induced by a high-uric acid diet / potassium oxonate after intravenous injection of a uricase mRNA composition. The dashed line in the figure represents the upper limit of normal serum uric acid levels, 360 μmol / L. A serum uric acid level below 360 μmol / L is considered normal. That is, after administration, a serum uric acid level below 360 μmol / L is considered effective, while a level above 360 ​​μmol / L is considered ineffective. Figure 7b shows the serum urate oxidase activity in rats with hyperuricemia induced by a high-uric acid diet / potassium oxonate after intravenous injection of a uricase mRNA composition. The uricase mRNA composition in the figure is uricase mRNA encapsulated in LNP formulation 2. The dosage indicated in the figure is the mRNA dosage in the composition.

[0036] Figure 8a shows the changes in serum uric acid levels in UOX-KO hyperuricemia mice after intravenous injection of a urate oxidase mRNA composition. The dotted line in the figure represents the upper limit of normal serum uric acid levels of 360 μmol / L. Serum uric acid below 360 μmol / l is considered normal, that is, after administration, the animal's serum uric acid is below 360 μmol / l, which means the drug is considered to be effective, and above 360 ​​μmol / l, the drug is considered to be ineffective or ineffective. Figure 8b shows the serum urate oxidase activity in UOX-KO hyperuricemia mice after intravenous injection of a urate oxidase mRNA composition. The urate oxidase mRNA composition in the figure is urate oxidase mRNA encapsulated in LNP formulation 2. The dose in the figure is the dose of mRNA in the composition.

[0037] Figure 9a shows the changes in serum uric acid levels in UOX-KO hyperuricemia mice after intravenous injection of a urate oxidase mRNA composition. The dotted line in the figure represents the upper limit of normal serum uric acid levels of 360 μmol / L. Serum uric acid below 360 μmol / l is considered normal, that is, after administration, the animal's serum uric acid is below 360 μmol / l, which means the drug is considered to be effective, and above 360 ​​μmol / l, the drug is considered to be ineffective or ineffective. Figure 9b shows the serum urate oxidase activity in UOX-KO hyperuricemia mice after intravenous injection of a urate oxidase mRNA composition. The urate oxidase mRNA composition in the figure is urate oxidase mRNA encapsulated in LNP formulation 2. The dosage in the figure is the dosage of mRNA in the composition.

[0038] Figure 10 shows serum uricase activity in normal rats after intravenous injection of uricase protein or a uricase mRNA composition. The uricase mRNA composition in the figure is uricase mRNA encapsulated in LNP formulation 2. The dosage of the uricase mRNA composition in the figure is the dosage of the mRNA in the composition.

[0039] Figure 11 shows serum uric acid levels in normal rats after intravenous injection of uricase protein or a uricase mRNA composition. The uricase mRNA composition in the figure is uricase mRNA encapsulated in LNP formulation 2. The dosage of the uricase mRNA composition in the figure is the dosage of the mRNA in the composition.

[0040] Figure 12a shows the serum uricase activity in normal mice after intravenous injection of a uricase mRNA composition. Figure 12b shows the serum uric acid levels in normal mice after intravenous injection of a uricase mRNA composition. The uricase mRNA composition in the figure is uricase mRNA encapsulated in LNP formulation 2. The dosage of the uricase mRNA composition in the figure represents the dosage of the mRNA in the composition.

[0041] FIG13a shows the serum uricase activity in normal rats after intravenous injection of a uricase mRNA composition with extended half-life.

[0042] Figure 13b shows serum uric acid levels in normal rats after intravenous injection of a uricase mRNA composition with an extended half-life. The uricase mRNA composition in the figure is uricase mRNA encapsulated in LNP formulation 2. The dosage of the uricase mRNA composition in the figure is the dosage of the mRNA in the composition. DETAILED DESCRIPTION

[0043] Provided herein are nucleic acid molecules comprising an open reading frame (ORF) encoding urate oxidase, a functional fragment or variant thereof. Also provided herein are compositions comprising the nucleic acid molecules, including lipid nanoparticles (LNPs), and related methods and uses for treating or preventing gout, gouty arthritis, hyperuricemia, or tumor lysis syndrome.

[0044] 1. General Technology

[0045] The techniques and procedures described or referenced herein include those generally well understood and / or commonly employed by those skilled in the art using conventional methods, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Current Protocols in Molecular Biology (Ausubel et al., eds., 2003).

[0046] 2. Terminology

[0047] Unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For the purpose of interpreting this specification, the following terminology will be applied, and where appropriate, terms used in the singular will also include the plural form, and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. If any description of a term set forth conflicts with any document incorporated herein by reference, the terminology set forth below shall prevail.

[0048] As used herein and unless otherwise indicated, the term "lipid" refers to a group of organic compounds that include, but are not limited to, fatty acid esters and are generally characterized by being poorly soluble in water but soluble in many non-polar organic solvents. Although lipids generally have poor water solubility, certain classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG2000) have limited water solubility and are soluble in water under certain conditions. Known lipid types include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be divided into at least three categories: (1) "simple lipids," including fats and oils, as well as waxes; (2) "compound lipids," including phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) "derivatized lipids," such as steroids. In addition, as used herein, lipids also include lipid-like compounds. The term "lipid-like compound," also referred to as "lipid-like," refers to lipid-like compounds (e.g., amphiphilic compounds with lipid-like physical properties).

[0049] The term "lipid nanoparticle" or "LNP" refers to a particle with at least one nanometer (nm) size (e.g., 1 to 1,000 nm) containing one or more types of lipid molecules. The LNP provided herein may further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule partially or completely encapsulated in a lipid shell. Specifically, in some embodiments, the payload is a negatively charged molecule (e.g., mRNA encoding uricase), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is expected that the cationic lipid can interact with the negatively charged payload molecule and promote the payload to be incorporated into and / or be encapsulated in the LNP during LNP formation. Other lipids that can form a part for LNP as provided herein include, but are not limited to, neutral lipids and charged lipids, such as steroids, polymer-conjugated lipids and various zwitterionic lipids.

[0050] The term "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or can be positively charged in response to the pH value or hydrogen ion activity of its environment (e.g., the environment of its intended use). Therefore, the term "cationic" encompasses both "permanent cations" and "cationizable". In certain embodiments, the positive charge in the cationic lipid is derived from the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that is positively charged in the environment of its intended use (e.g., at physiological pH).

[0051] The term "polymer-conjugated lipid" refers to a molecule that comprises both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a pegylated lipid (PEG-lipid), wherein the polymer portion comprises polyethylene glycol.

[0052] The term "neutral lipid" encompasses any lipid molecule that exists in an uncharged form or in a neutral zwitterionic form at a selected pH value or within a selected pH range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment of the intended lipid use, such as a physiological pH value. As non-limiting examples, neutral lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); phosphatidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl))dimethylammonio)ethyl hydrogenphosphate (DOCP); sphingomyelin (SM); ceramides; steroids, such as sterols and their derivatives. The neutral lipids provided herein can be synthetic or derived from (isolated or modified from) natural sources or compounds.

[0053] The term "charged lipid" encompasses any lipid molecule that exists in a positively or negatively charged form at a selected pH value or within a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH conditions in the environment of the intended lipid use, such as physiological pH. As a non-limiting example, the neutral lipids that can be used in conjunction with the present disclosure include but are not limited to phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-racemic-glycerol) sodium salt (DOPG-Na) and 1,2-dioleoyl-sn-glycero-3-phospho-sodium salt (DOPA-Na). The charged lipids provided herein can be synthetic or derived from (isolated or modified from) natural sources or compounds.

[0054] As used herein, "lipoplex (LPX)" generally refers to a complex formed spontaneously by lipids (cationic lipids), for example, a complex formed by a cationic lipid and a negative nucleic acid (eg, mRNA).

[0055] As used herein, the terms "signal peptide" and "signal sequence" can be used interchangeably and refer to sequences that can direct protein transport or extracellular export. The term encompasses signal sequence polypeptides and nucleic acid sequences encoding the signal sequence. Therefore, reference to a signal sequence in the context of nucleic acids actually refers to a nucleic acid sequence encoding a signal sequence polypeptide. In some embodiments, an mRNA has an ORF encoding a signal peptide fused to uricase. A signal peptide comprising 15-60 amino acids at the N-terminus of a protein is typically required for transmembrane translocation on the secretory pathway, and therefore, generally controls the entry of most proteins into the secretory pathway in eukaryotes and prokaryotes. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides ribosomes to the rough endoplasmic reticulum (ER) membrane, and the growing peptide chain is transported through it for processing. ER processing produces a mature protein, wherein the signal peptide is typically cut from the precursor protein by the ER-resident signal peptidase of the host cell, or it remains uncut and acts as a membrane anchor. Signal peptides can also promote protein targeting to the cell membrane. In some embodiments, the signal peptide can be 15-60 amino acids in length, and the corresponding signal sequence can be 45-180 nucleotides in length. Signal peptides from heterologous genes that regulate the expression of genes other than uricase are known in the art and can be tested for their desired properties before incorporation into the nucleic acids of the invention.

[0056] As used herein, the term "peroxisome targeting sequence" refers to a sequence that can direct protein transport targeting to the peroxisome. The term encompasses peroxisome targeting sequence polypeptides and nucleic acid sequences encoding the targeting sequence. In some embodiments, the therapeutic nucleic acids described herein, such as mRNA, have an ORF encoding a peroxidase targeting sequence fused to uricase. As used herein, the peroxisome targeting sequence may be at the N-terminus of the protein and / or mRNA sequence, or at the C-terminus of the protein and / or mRNA sequence, wherein the peroxisome targeting sequence at the C-terminus is generally 3 amino acids, and the corresponding nucleic acid length is generally 9 amino acids. Peroxisome targeting sequences from heterologous genes are known in the art and can be tested for their desired properties and then incorporated into the nucleic acids of the present invention.

[0057] As used herein, the term mRNA, also known as messenger RNA, generally refers to a type of single-stranded RNA that is transcribed from a DNA template, carries genetic information, and guides protein synthesis. Once mRNA is transcribed from DNA according to the Watson-Crick base pairing principle, it contains base sequences corresponding to certain functional segments of the DNA molecule, serving as a direct template for protein biosynthesis. As used herein, mRNA includes a nucleic acid molecule capable of encoding uricase, that is, at least a portion of the mRNA is capable of encoding uricase.

[0058] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary animals.

[0059] The term "composition" is intended to encompass a product containing specified ingredients (eg, mRNA molecules provided herein), optionally in specified amounts.

[0060] As used interchangeably herein, the terms "polynucleotide" or "nucleic acid" refer to nucleotide polymers of any length, and include, for example, DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. Nucleic acids can be in single-stranded or double-stranded form. As used herein and unless otherwise indicated, "nucleic acid" also includes nucleic acid mimics, such as locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholino nucleic acids. As used herein, "oligonucleotide" refers to a short synthetic polynucleotide, generally but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Unless otherwise indicated, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5′ to 3′ addition of the nascent RNA transcript is called the transcription direction; the sequence region on the DNA chain that has the same sequence as the RNA transcript and is located at the 5′ end relative to the 5′ end of the RNA transcript is called the “upstream sequence”; the sequence region on the DNA chain that has the same sequence as the RNA transcript and is located at the 3′ end relative to the 3′ end of the RNA transcript is called the “downstream sequence”.

[0061] "Isolated nucleic acid" refers to a nucleic acid, such as RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences and proteins or complexes (such as ribosomes and polymerases) that naturally accompany the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. In addition, when prepared by recombinant techniques, an "isolated" nucleic acid molecule, such as an mRNA molecule, can be substantially free of other cellular material or culture medium, or when chemically synthesized, it can be substantially free of chemical precursors or other chemicals. In specific embodiments, one or more nucleic acid molecules encoding uricase described herein are isolated or purified. The term includes nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA or RNA isolates, as well as chemically synthesized analogs or analogs biologically synthesized by heterologous systems. Substantially pure molecules can include isolated forms of the molecule.

[0062] The term "coding nucleic acid" or its grammatical equivalents, when used to refer to a nucleic acid molecule, includes (a) a nucleic acid molecule that can be transcribed to produce mRNA and subsequently translated into peptides and / or polypeptides when in its natural state or manipulated by methods well known to those skilled in the art; and (b) the mRNA molecule itself. The antisense strand is the complementary sequence of such a nucleic acid molecule, and the coding sequence can be inferred therefrom. The term "coding region" refers to the portion of a coding nucleic acid sequence that is translated into a peptide or polypeptide. The term "untranslated region" or "UTR" refers to the portion of a coding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of the UTR relative to the coding region of the nucleic acid molecule, the UTR is referred to as a 5'-UTR if it is located at the 5' end of the coding region, and the UTR is referred to as a 3'-UTR if it is located at the 3' end of the coding region.

[0063] The term "corresponding DNA sequence" or its grammatical equivalents when used in relation to an RNA sequence means the DNA sequence from which the RNA is transcribed. For example, the corresponding DNA sequence of the RNA sequence GCUGGAGCCUCGGUGGC is GCTGGAGCCTCGGTGGC.

[0064] As used herein, an "open reading frame (ORF)" is a nucleotide sequence that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TGA or TAG), and encodes a polypeptide. It should be understood that an open reading frame (ORF) as used herein includes both DNA and RNA sequences.

[0065] As used herein, the term codon optimization refers to replacing one, at least one, or more than one codon in a parent polypeptide encoding nucleic acid with codons encoding the same amino acid residue with different relative usage frequencies in the cell. In some embodiments, the mRNA described herein comprises a codon-optimized nucleic acid sequence, wherein the open reading frame (ORF) with the codon-optimized nucleic acid sequence encodes a uricase protein sequence. Methods for codon optimization are known in the art. In some embodiments, codon optimization can be used to match codon frequencies in the target and host organisms to ensure correct folding; bias toward GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcription and translation control regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites in the encoded protein; add, remove, or reorganize protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translation rates to ensure that each structure of the protein is correctly folded; or reduce or eliminate problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art. In some embodiments, ORF sequences are optimized using an optimization algorithm.

[0066] As used herein, the term "mRNA" refers to a messenger RNA molecule comprising one or more open reading frames (ORFs), which can be translated by a cell or organism having the mRNA to produce one or more peptide or protein products. The region containing one or more ORFs is referred to as the coding region of the mRNA molecule. In certain embodiments, the mRNA molecule further comprises one or more untranslated regions (UTRs).

[0067] The term "nucleobase" encompasses purines and pyrimidines, including the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural or synthetic analogs or derivatives thereof.

[0068] As used herein, the term "functional nucleotide analogue" refers to a modified form of a classical nucleotide A, G, C, U, or T that (a) retains the base pairing properties of the corresponding classical nucleotide and (b) contains at least one chemical modification of the (i) nucleobase, (ii) sugar group, (iii) phosphate group, or (iv) any combination of (i) to (iii) of the corresponding natural nucleotide. As used herein, base pairing encompasses not only the classical Watson-Crick adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between a classical nucleotide and a functional nucleotide analogue, or between a pair of functional nucleotide analogues, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonds to be formed between the modified nucleobase and the classical nucleobase, or between two complementary modified nucleobase structures. For example, a functional analogue of guanosine (G) retains the ability to base pair with cytosine (C) or a functional analogue of cytosine. An example of such non-classical base pairing is base pairing between a modified nucleotide inosine and adenine, cytosine, or uracil. As described herein, functional nucleotide analogs can be naturally occurring or non-naturally occurring. Thus, nucleic acid molecules containing functional nucleotide analogs can have at least one modified nucleobase, sugar group, and / or internucleoside bond. Provided herein are chemical modifications of the nucleobases, sugar groups, or internucleoside bonds of nucleic acid molecules.

[0069] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer having more than fifty (50) amino acid residues linked by covalent peptide bonds. That is, a description of a polypeptide equally applies to a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog). As used herein, the terms encompass amino acid chains of any length, including full-length proteins (e.g., uricase).

[0070] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules, or two or more nucleic acid molecules, as determined by sequence alignment." amino acid sequence identity percentage (%) " is defined as the percentage of the identical amino acid residues in the candidate sequence and the reference polypeptide sequence relative to the reference polypeptide sequence, after alignment and introduction of a gap factor. If necessary, maximum sequence identity percentage can be achieved and any conservative substitutions are not considered as part of sequence identity. The comparison for determining amino acid sequence identity percentage can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0071] "Modification" of an amino acid residue / position refers to an alteration of the primary amino acid sequence compared to the starting amino acid sequence, wherein the alteration is caused by a sequence change involving the amino acid residue / position. For example, typical modifications include replacing a residue with another amino acid (e.g., conservative or non-conservative substitution), inserting one or more (e.g., typically less than 5, 4, or 3) amino acids / positions near the residue, and / or deleting the residue / position.

[0072] The term "vector" refers to a substance used to carry or include a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a uricase described herein, so that the nucleic acid sequence can be introduced into a host cell or used as a transcription template for in vitro transcription reactions in a cell-free system to produce mRNA. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include a selection sequence or marker for stable integration into the host cell chromosome. In addition, the vector can include one or more selective marker genes and appropriate transcription or translation control sequences. The included selective marker gene can, for example, provide resistance to antibiotics or toxins, supplement nutrient deficiencies, or provide key nutrients not present in the culture medium. Transcription or translation control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules (e.g., nucleic acid molecules encoding two or more different uricases) are co-transcribed or co-translated, the two nucleic acid molecules can be inserted into the same expression vector or into separate expression vectors. For single vector transcription and / or translation, the encoding nucleic acid can be operably linked to a common transcription or translation control sequence, or to different transcription or translation control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art can be used to confirm that the nucleic acid molecule is introduced into the host cell. Such methods include: utilizing nucleic acid analysis such as Northern blot or polymerase chain reaction (PCR) amplification, immunoblotting for gene product expression or other suitable analytical methods to detect the introduced nucleic acid sequence or its corresponding expressed gene product. It will be understood by those skilled in the art that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product (e.g., mRNA transcript of the nucleic acid as described herein), and it will also be understood that expression levels can be optimized by methods well known in the art to obtain sufficient expression products.

[0073] The term "administer" or "administration" refers to the operation of injecting or otherwise physically delivering a substance (e.g., a lipid nanoparticle composition described herein) present in vitro into a patient's body, such as by transmucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When treating a disease, disorder, condition, or symptom thereof, the administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom thereof. When preventing a disease, disorder, condition, or symptom thereof, the administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom thereof.

[0074] "Long-term" administration, as opposed to acute mode, refers to administration of one or more doses in a continuous mode (e.g., over a period of time, such as days, weeks, months, or years), thereby maintaining the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration means that the treatment is not continued uninterrupted, but is cyclical in nature.

[0075] As used herein, the term "targeted delivery" or verb form "targeting" refers to a process in which the delivered agent (the therapeutic nucleic acid such as mRNA in the lipid nanoparticle composition as described herein) is promoted to reach a specific organ, tissue, cell and / or intracellular compartment (referred to as a target location) compared to delivery to any other organ, tissue, cell or intracellular compartment (referred to as a non-target location). Targeted delivery can be detected using methods known in the art, for example, by comparing the concentration of the delivered agent in the target cell population with the concentration of the delivered agent at the non-target cell population after systemic administration. In certain embodiments, targeted delivery makes the concentration at the target location at least 2 times higher than the concentration at the non-target location.

[0076] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms; eliminate symptoms and / or potential causes; prevent the occurrence of symptoms and / or their potential causes; and / or improve or remedy damage caused by or associated with a disease, disorder, or condition. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0077] As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., a therapeutic nucleic acid such as mRNA or pharmaceutical composition as described herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder or condition, and / or its associated symptoms. A "therapeutically effective amount" of an agent of the present disclosure (e.g., a lipid nanoparticle composition as described herein) may vary according to a number of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount comprises an amount in which any toxic or deleterious effects of the agent are outweighed by the therapeutically beneficial effects of the agent. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a lipid nanoparticle composition as described herein or a therapeutic or prophylactic agent (e.g., therapeutic mRNA) contained therein that is effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0078] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended preventive effect, such as preventing a disease, disorder, condition, or related symptoms (e.g., gout, gouty arthritis, hyperuricemia, or tumor lysis syndrome, or symptoms caused thereby), delaying its onset (or recurrence), or reducing the likelihood of its onset (or recurrence). Typically, but not necessarily, a prophylactic dose is used for a subject before a disease, disorder, or condition, or in its early stages, so the prophylactic effective amount may be less than the therapeutically effective amount. A complete therapeutic or preventive effect may not occur by administering a single dose, but may only occur after administering a series of doses. Therefore, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0079] The terms "prevent," "preventing," or "prevention" refer to reducing the likelihood of onset (or recurrence) of a disease, disorder, condition, or associated symptoms (e.g., gout, gouty arthritis, hyperuricemia, or tumor lysis syndrome, or symptoms caused thereby).

[0080] The term "prophylactic agent" refers to any agent that can completely or partially inhibit the development, recurrence, onset, or spread of a disease and / or its associated symptoms in a subject.

[0081] The term "therapeutic agent" refers to any agent useful in treating, preventing, or alleviating a disease, disorder, or condition, including any agent useful in treating, preventing, or alleviating one or more symptoms of a disease, disorder, or condition and / or its associated symptoms.

[0082] The term "therapy" refers to any regimen, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease, disorder, or condition. In certain embodiments, the term "therapies" or "therapy" refers to biological therapies, supportive therapies, and / or other therapies known to those skilled in the art, such as medical personnel, that can be used to prevent, manage, treat, and / or improve a disease, disorder, or condition.

[0083] The terms "subject" and "patient" are used interchangeably. As used herein, in certain embodiments, the subject is a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey and a human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing gout, gouty arthritis, hyperuricemia, or tumor lysis syndrome.

[0084] By "substantially all" is meant at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0085] As used herein and unless otherwise indicated, the term "about" or "approximately" means an acceptable error for a particular value determined by one of ordinary skill in the art, which depends in part on the manner in which the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, within 0.5%, within 0.05%, or less of a given value or range.

[0086] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0087] All publications, patent applications, and other references cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The publications discussed herein provide their disclosures solely as of the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed.

[0088] A number of embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, the descriptions in the experimental section and examples are intended to illustrate, but not to limit, the scope of the invention described in the claims.

[0089] 3. Therapeutic nucleic acids

[0090] 3.1 Open Reading Frame (ORF)

[0091] In one aspect, provided herein are therapeutic nucleic acid molecules for treating or preventing gout, gouty arthritis, hyperuricemia, or tumor lysis syndrome. In some embodiments, the therapeutic nucleic acid comprises an open reading frame (ORF) encoding uricase, its functional fragment, or variant, which, after being administered to a subject in need thereof, is expressed by cells in the subject to produce the encoded uricase, its functional fragment, or variant. In some embodiments, the therapeutic nucleic acid molecule is a DNA molecule. In other embodiments, the therapeutic nucleic acid molecule is an RNA molecule. In specific embodiments, the therapeutic nucleic acid molecule is an mRNA molecule. In some embodiments, the nucleic acid or therapeutic nucleic acid molecule is a non-naturally occurring nucleic acid or therapeutic nucleic acid molecule.

[0092] In some embodiments, the uricase, functional fragment, or variant thereof encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the uricase, functional fragment, or variant thereof encoded by the mRNA payload can have a therapeutic effect when expressed in a cell.

[0093] In some embodiments, the nucleic acid molecules of the present disclosure include mRNA molecules. In specific embodiments, the nucleic acid molecules include at least one coding region (e.g., open reading frame (ORF)) encoding a protein of interest. In some embodiments, the nucleic acid molecules further include at least one untranslated region (UTR). In specific embodiments, the untranslated region (UTR) is located upstream (5' end) of the coding region and is referred to herein as a 5'-UTR. In specific embodiments, the untranslated region (UTR) is located downstream (3' end) of the coding region and is referred to herein as a 3'-UTR. In some embodiments, the nucleic acid molecules include both a 5'-UTR and a 3'-UTR. In some embodiments, the 5'-UTR includes a 5'-cap structure. In some embodiments, the nucleic acid molecules include a Kozak sequence (e.g., in a 5'-UTR). In some embodiments, the nucleic acid molecules include a poly-A region (e.g., in a 3'-UTR). In some embodiments, the nucleic acid molecules include a polyadenylation signal (e.g., in a 3'-UTR). In some embodiments, the nucleic acid molecules include a stabilizing region (e.g., in a 3'-UTR). In some embodiments, the nucleic acid molecules include a secondary structure. In some embodiments, the secondary structure is a stem-loop. In some embodiments, the nucleic acid molecule comprises a stem-loop sequence (e.g., in a 5′-UTR and / or a 3′-UTR). In some embodiments, the nucleic acid molecule comprises one or more intronic regions that can be excised during splicing. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from a 5′-UTR and a coding region. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from a coding region and a 3′-UTR. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from a 5′-UTR, a coding region, and a 3′-UTR.

[0094] In some embodiments, the uricase is a full-length uricase. In some other embodiments, the functional fragment or variant of the uricase is a truncated uricase or a uricase fusion protein that retains uricase activity.

[0095] As used herein, the uricase is a homotetrameric enzyme composed of four identical 34 kDa subunits that catalyzes the oxidation of uric acid. The oxidation product is allantoin, which is more soluble and more easily excreted. Specifically, uricase catalyzes the reaction of uric acid with O2 and H2O to form 5-hydroxy-isourate (HIU) and the release of H2O2. HIU is an unstable product that can undergo non-enzymatic hydrolysis to generate 2-oxo-4-hydroxy-5-ureidoimidazole (OHCU) and then spontaneously decarboxylate to form racemic allantoin. Urate oxidase is found in many organisms (such as archaea, bacteria, and eukaryotes). However, many primates, including humans, do not express uricase due to nonsense mutations at codons 33 and 187 and mutations at the splice acceptor site in intron 2 during evolution. Due to the lack of urate oxidase expression in the human body, high blood uric acid levels can, in some cases, easily lead to hyperuricemia, gout, and tumor lysis syndrome. Gout is an inflammatory arthritis. Long-term serum uric acid levels above 6.8 mg / dL can cause urate crystals to precipitate in tissues and accumulate in joints, causing an acute inflammatory response. Tumor lysis syndrome (TLS) refers to a tumor emergency characterized by hyperuricemia, hyperkalemia, hyperphosphatemia, hypocalcemia, and acute renal failure caused by the massive lysis of tumor cells and the release of intracellular metabolites during treatment in cancer patients.

[0096] In some embodiments, the uricase can be selected from the following species: Aspergillus flavus, Arthritis Bacterium, Candida utilis, human pseudogene, porcine, baboon, monkey, bovine, canine, etc.

[0097] In some embodiments, the uricase can be any wild-type uricase.

[0098] In some embodiments, the uricase can be a functional fragment of any wild-type uricase.

[0099] In some embodiments, the uricase can be a full-length or truncated wild-type uricase, optionally in which one or more amino acids have been substituted.

[0100] In some embodiments, the uricase may be a fusion protein composed of functional fragments of uricases from different species.

[0101] In some embodiments, the uricase has an amino acid sequence as set forth in any one of SEQ ID NOs: 1-21, or at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to any one of SEQ ID NOs: 1-21.

[0102] In some embodiments, the uricase is derived from Arthrobacter and has an amino acid sequence as set forth in SEQ ID NO:4 or 5, or at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO:4 or 5.

[0103] In some embodiments, the uricase is derived from aflatoxin and has an amino acid sequence as set forth in SEQ ID NO:3, or at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:3.

[0104] In some embodiments, the uricase is derived from a human pseudogene and has an amino acid sequence as set forth in SEQ ID NO: 1 or at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 1.

[0105] In some embodiments, the uricase is derived from a mammal and has an amino acid sequence as set forth in SEQ ID NO:2, or at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:2.

[0106] In some embodiments, the uricase is derived from Candida utilis and has an amino acid sequence as set forth in SEQ ID NO: 9 or 10, or at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 9 or 10.

[0107] In some embodiments, the uricase is a truncated or full-length protein of uricase that does not contain any signal peptide or localization peptide. In some alternative embodiments, the uricase is a uricase fusion protein, which is a fusion protein of a signal peptide and an optional spacer sequence with uricase, or a fusion protein of a peroxisomal localization peptide with uricase. In some other embodiments, the uricase is a uricase fusion protein, which is a fusion protein comprising a signal peptide, a spacer sequence, and uricase from the N-terminus to the C-terminus. In some other embodiments, the uricase is a uricase fusion protein, which is a fusion protein comprising a signal peptide, a spacer sequence, a half-life extending element, an optional linker, and uricase from the N-terminus to the C-terminus, or a fusion protein comprising a signal peptide, a spacer sequence, uricase oxidase, an optional linker, and a half-life extending element from the N-terminus to the C-terminus. In some other embodiments, the uricase is a uricase fusion protein, which is a fusion protein comprising a signal peptide, a spacer sequence, a half-life extending element, a linker, and uricase from the N-terminus to the C-terminus, or a fusion protein comprising a signal peptide, a spacer sequence, uricase, a linker, and a half-life extending element from the N-terminus to the C-terminus.

[0108] In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 22-45, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 22-45.

[0109] In some embodiments, the spacer sequence comprises the amino acid sequence of any one of SEQ ID NOs: 50-53, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 50-53.

[0110] In some embodiments, the signal peptide and / or spacer sequence is fused to the N-terminus or C-terminus of the uricase.

[0111] In some embodiments, the half-life extending element comprises SEQ ID NO: 381 or 382, ​​or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 381 or 382.

[0112] In some embodiments, the linker comprises SEQ ID NO: 383, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 383.

[0113] In some embodiments, the peroxisome localization peptide comprises the amino acid sequence of any one of SEQ ID NOs: 46-49, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 46-49.

[0114] In some embodiments, the peroxisome targeting peptide is fused to the N-terminus or C-terminus of the uricase.

[0115] In some embodiments, the ORF is codon-optimized.

[0116] In some embodiments, the ORF is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 67-212, 386-389, or their corresponding DNA sequences.

[0117] In some embodiments, the therapeutic nucleic acid molecules of the present disclosure comprise an open reading frame (ORF) encoding uricase, a functional fragment or variant thereof, and the therapeutic nucleic acid molecule comprises the corresponding DNA sequence of any one of SEQ ID NOs: 67-212, 386-389. In specific embodiments, the therapeutic nucleic acid molecule is a DNA molecule. In other embodiments, the therapeutic nucleic acid molecules of the present disclosure comprise an open reading frame (ORF) encoding uricase, a functional fragment or variant thereof, and the therapeutic nucleic acid molecule comprises any one of SEQ ID NOs: 67-212, 386-389. In specific embodiments, the therapeutic nucleic acid molecule is an mRNA molecule.

[0118] 3.2 5'-cap structure

[0119] Without being bound by theory, it is expected that the 5′-cap structure of the polynucleotide participates in nuclear export and increases polynucleotide stability, and binds to mRNA cap binding protein (CBP), which is responsible for polynucleotide stability in the cell and causes translational competence through the formation of mature circular mRNA species through the association of CBP with poly-A binding protein. The 5′-cap structure further facilitates the removal of 5′-proximal introns during mRNA splicing. Therefore, in some embodiments, the nucleic acid molecules of the present disclosure include a 5′-cap structure.

[0120] Nucleic acid molecules can be capped at the 5' end by the cell's endogenous transcriptional machinery, thereby generating a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue of the polynucleotide and the sense nucleotide transcribed at the 5' end. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides at the 5' end of the polynucleotide can also be optionally 2'-O-methylated. 5'-Decapping via hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0121] In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more alterations to the native 5'-cap structure produced by endogenous processes. Without being bound by theory, modifications to the 5'-cap can increase the stability of the polynucleotide, increase the half-life of the polynucleotide, and increase the translation efficiency of the polynucleotide.

[0122] Exemplary changes to the natural 5'-cap structure include creating a non-hydrolyzable cap structure to prevent decapping and thereby increase the half-life of the polynucleotide. In some embodiments, because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester bond, in some embodiments, modified nucleotides may be used during the capping reaction. For example, in some embodiments, a vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) may be used for α-thioguanosine nucleotides to create a phosphorothioate bond in the 5'-ppp-5' cap according to the manufacturer's instructions. Additional modified guanosine nucleotides, such as α-methylphosphonic acid and selenophosphate nucleotides, may be used.

[0123] Additional exemplary alterations to the native 5′-cap structure also include modifications at the 2′ and / or 3′ position of the capped guanosine triphosphate (GTP), replacement of the sugar ring oxygen (resulting in the oxygen of the carbocyclic ring) with a methylene moiety (CH2), modifications at the triphosphate bridge portion of the cap structure, or modifications at the nucleobase (G) portion.

[0124] Additional exemplary changes to the natural 5'-cap structure include, but are not limited to, 2'-O-methylation of the ribose sugar at the 5'-terminus of the polynucleotide and / or the 5'-terminal nucleotide on the sugar 2'-hydroxyl group (as described above). A plurality of different 5'-cap structures can be used to produce the 5'-cap of a polynucleotide (e.g., an mRNA molecule). Additional exemplary 5'-cap structures that can be used in conjunction with the present disclosure further include those described in International Patent Publications WO2008127688, WO 2008016473, and WO 2011015347, the entire contents of which are incorporated herein by reference.

[0125] In various embodiments, the 5'-terminal cap may comprise a cap analog. Cap analogs are also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, whose chemical structure differs from that of a natural (i.e., endogenous, wild-type, or physiological) 5'-cap while retaining cap function. Cap analogs can be synthesized and / or attached to a polynucleotide chemically (i.e., non-enzymatically) or enzymatically.

[0126] For example, the anti-reverse cap analog (ARCA) cap contains two guanosines linked via a 5′-5′-triphosphate group, wherein one of the guanosines contains an N7-methyl group as well as a 3′-O-methyl group (i.e., N7, 3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, i.e., m7G-3′mppp-G, which can be equivalently referred to as 3′O-Me-m7G(5′)ppp(5′)G). The 3′-O atom of the other, unchanged guanosine is linked to the 5′-terminal nucleotide of the capped polynucleotide (e.g., mRNA). The N7- and 3′-O-methylated guanosines provide the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary cap structure is mCAP, which is similar to ARCA but has a 2′-O-methyl group on the guanosine (i.e., N7, 2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, i.e., m7Gm-ppp-G).

[0127] In some embodiments, the cap analog can be a dinucleotide cap analog. As a non-limiting example, a dinucleotide cap analog can be modified with a boranophosphate or a phophoroselenoate at different phosphate positions, such as the dinucleotide cap analogs described in U.S. Patent No. 8,519,110, the entire contents of which are incorporated herein by reference in their entirety.

[0128] In some embodiments, the cap analog can be an N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, e.g., Kore et al., Bioorganic & Medicinal Chemistry 2013 21:4570-4574 for various cap analogs and methods for synthesizing cap analogs; the entire contents of which are incorporated herein by reference). In other embodiments, the cap analog that can be used in conjunction with the nucleic acid molecules of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0129] In various embodiments, the cap analog may comprise a guanosine analog. Useful guanosine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0130] Without being bound by theory, it is expected that although cap analogs allow for simultaneous capping of polynucleotides in in vitro transcription reactions, up to 20% of transcripts remain uncapped. This, along with the structural differences between cap analogs and the natural 5′-cap structure of polynucleotides produced by the cell's endogenous transcription machinery, may lead to reduced translational capacity and cellular stability.

[0131] Therefore, in some embodiments, the nucleic acid molecules of the present disclosure can also be capped after transcription using enzymes to produce a more authentic 5′-cap structure. As used herein, the phrase “more authentic” refers to a feature that closely reflects or mimics an endogenous or wild-type feature in structure or function. That is, a “more authentic” feature better represents endogenous, wild-type, natural or physiological cellular function and / or structure than a synthetic feature or analog of the prior art, or it outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more aspects. Non-limiting examples of more authentic 5′-cap structures that can be used in conjunction with the nucleic acid molecules of the present disclosure are structures that have, in particular, enhanced binding to cap-binding proteins, increased half-life, reduced sensitivity to 5′-endonucleases and / or reduced 5′-decapping compared to synthetic 5′-cap structures known in the art (or compared to wild-type, natural or physiological 5′-cap structures). For example, in some embodiments, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can generate a classic 5'-5'-triphosphate bond between the 5'-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, wherein the cap guanosine contains an N7-methylation and the 5'-terminal nucleotide of the polynucleotide contains a 2'-O-methyl group. This structure is referred to as a cap 1 structure. Compared to other 5' cap analog structures known in the art, for example, this cap results in higher translational capacity, cellular stability, and reduced activation of cellular proinflammatory cytokines. Other exemplary cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')-ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).

[0132] In some embodiments, the 5'-cap structure used herein is selected from m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7GpppN, m7GpppNmpNp, m7GpppNmpNmp. In a preferred embodiment, the 5'-cap structure used herein is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0133] Without being bound by theory, it is contemplated that the nucleic acid molecules of the present disclosure can be capped post-transcriptionally, and because this method is relatively efficient, nearly 100% of the nucleic acid molecules can be capped.

[0134] 3.3 Untranslated Region (UTR)

[0135] In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more untranslated regions (UTRs). In some embodiments, the UTR is located upstream of the coding region in the nucleic acid molecule and is referred to as a 5′-UTR. In some embodiments, the UTR is located downstream of the coding region in the nucleic acid molecule and is referred to as a 3′-UTR. The sequence of the UTR may be homologous or heterologous to the sequence of the coding region found in the nucleic acid molecule. Multiple UTRs may be included in the nucleic acid molecule and may have the same or different sequences and / or gene origins. According to the present disclosure, any portion of the UTR in the nucleic acid molecule (including without any portion) may be optimized, and any portion may independently contain one or more different structures or chemical modifications before and / or after optimization.

[0136] In some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise UTRs and coding regions that are homologous to each other. In other embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise UTRs and coding regions that are heterologous to each other. In some embodiments, to monitor the activity of UTR sequences, nucleic acid molecules comprising coding sequences of UTRs and detectable probes can be administered in vitro (e.g., in cell or tissue culture) or in vivo (e.g., to a subject), and the effects of the UTR sequences (e.g., regulating expression levels, cellular localization of the encoded product, or half-life of the encoded product) can be measured using methods known in the art.

[0137] In some embodiments, the nucleic acid molecules of the present disclosure comprise a 5′-UTR selected from SEQ ID NOs: 54-57, 384, or their corresponding DNA sequences. In some embodiments, the nucleic acid molecules of the present disclosure comprise a 3′-UTR selected from SEQ ID NOs: 58-62, 385, or their corresponding DNA sequences. In some embodiments, the nucleic acid molecules of the present disclosure comprise a 5′-UTR selected from SEQ ID NOs: 54-57, 384, or their corresponding DNA sequences and a 3′-UTR selected from SEQ ID NOs: 58-62, 385, or their corresponding DNA sequences. In further embodiments, the nucleic acid molecules may further comprise an ORF of any one of SEQ ID NOs: 67-212, 386-389, or their corresponding DNA sequences. In specific embodiments, the nucleic acid molecules described herein may be in vitro transcribed RNA molecules.

[0138] 3.4 Polyadenylation (Poly-A) Region

[0139] During natural RNA processing, long chains of adenosine nucleotides (poly-A regions) are typically added to messenger RNA (mRNA) molecules to increase the molecule's stability. Immediately after transcription, the 3'-end of the transcript is cleaved to release the 3'-hydroxyl group. Subsequently, poly-A polymerase adds a string of adenosine nucleotides to the RNA. This process, called polyadenylation, adds a poly-A region between 100 and 250 residues in length. Without being bound by theory, it is contemplated that poly-A regions may confer a number of advantages to the nucleic acid molecules of the present disclosure.

[0140] Therefore, in some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise a polyadenylation signal. In some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise one or more polyadenylation (poly-A) regions. In some embodiments, the poly-A region is entirely composed of adenine nucleotides or functional analogs thereof. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 3' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5' end and at least one poly-A region at its 3' end.

[0141] According to the present disclosure, in different embodiments, the poly-A region may have different lengths. Specifically, in some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 30 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 35 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 40 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 45 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 50 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 55 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 60 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 65 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 70 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 75 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 80 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 85 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 90 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 95 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 100 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 110 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 120 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 130 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 140 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 150 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 160 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 170 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 180 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 190 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 200 nucleotides in length.In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 225 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 250 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 275 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 300 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 350 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 400 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 450 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 500 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 600 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 700 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 800 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 900 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 1000 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is 1000 to 3000 nucleotides in length.

[0142] In some embodiments, the length of the poly-A region in a nucleic acid molecule can be selected based on the total length of the nucleic acid molecule or a portion thereof (e.g., the length of the coding region of the nucleic acid molecule or the length of the open reading frame, etc.). For example, in some embodiments, the poly-A region comprises about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the total length of the nucleic acid molecule containing the poly-A region.

[0143] In some embodiments, the 3'-poly(A) sequence comprises 10 to 400 adenosine nucleotides, preferably 50 to 400 adenosine nucleotides or 10 to 300 adenosine nucleotides; more preferably 50 to 250 adenosine nucleotides. Optionally, the 3'-poly(A) sequence comprises an adenosine polynucleotide and a linker sequence inserted therein, wherein the linker sequence is used to separate the adenine nucleotide sequence.

[0144] Without being bound by theory, it is expected that certain RNA binding proteins can bind to the poly-A region located at the 3' end of the mRNA molecule. These poly-A binding proteins (PABPs) can regulate mRNA expression, for example, by interacting with the translation initiation machinery in the cell and / or protecting the 3'-poly-A tail from degradation. Therefore, in some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure contain at least one binding site for a poly-A binding protein (PABP). In other embodiments, the nucleic acid molecule is bound to the PABP before being loaded into a delivery vehicle (e.g., a lipid nanoparticle). Typically, poly-A (e.g., a 3'-poly(A) sequence) is a continuous sequence of adenosine nucleotides. In some alternative embodiments, the poly-A (e.g., a 3'-poly(A) sequence) comprises an adenosine polynucleotide and a linker sequence inserted therein to separate the adenine nucleotide sequence.

[0145] In some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise poly-AG quadruplexes. A G quadruplex is a circular array of four hydrogen-bonded guanosine nucleotides that can be formed by G-rich sequences in DNA and RNA. In this embodiment, the G quadruplex is incorporated into one end of the poly-A region. The stability, protein yield, and other parameters of the resulting polynucleotide (e.g., mRNA) can be analyzed, including half-life at different time points. It has been found that the protein yield of the poly-AG quadruplex structure is at least 75% of the protein yield observed using only a poly-A region containing 120 nucleotides.

[0146] In some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure may include a poly-A region and may be stabilized by adding a 3′-stabilizing region. In some embodiments, the 3′-stabilizing region that can be used to stabilize nucleic acid molecules (e.g., mRNA) includes a poly-A or poly-AG quadruplex structure as described in International Patent Publication No. WO2013 / 103659, the contents of which are incorporated herein by reference in their entirety.

[0147] In other embodiments, 3′-stabilizing regions that can be used in conjunction with the nucleic acid molecules of the present disclosure include chain-terminating nucleosides such as, but not limited to, 3′-deoxyadenosine (cordycepin); 3′-deoxyuridine; 3′-deoxycytosine; 3′-deoxyguanosine; 3′-deoxythymidine; 2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, 2′,3′-dideoxythymidine; 2′-deoxynucleosides; or O-methyl nucleosides: 3′-deoxynucleosides; 2′,3′-dideoxynucleosides; 3′-O-methyl nucleosides; 3′-O-ethyl nucleosides; 3′-arabinoside, as well as other alternative nucleosides known in the art and / or described herein.

[0148] 3.5 Nucleobase Modification

[0149] In some embodiments, functional nucleotide analogs contain non-classical nucleobases. In some embodiments, the classical nucleobases in a nucleotide (e.g., adenine, guanine, uracil, thymine, and cytosine) can be modified or replaced to provide one or more functional analogs of the nucleotide. Exemplary modifications of nucleobases include, but are not limited to, one or more substitutions or modifications, including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkoxy, and / or thio substitutions; one or more fused rings or ring opening, oxidation, and / or reduction.

[0150] In some embodiments, the non-classical nucleobase is a modified uracil. Exemplary nucleobases and nucleosides with modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (s 2 U), 4-thio-uracil (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho 5 U), 5-aminoallyl-uracil, 5-halouracil (e.g., 5-iodouracil or 5-bromouracil), 3-methyluracil (m 3 U), 5-methoxyuracil (mo 5 U), uracil 5-oxyacetic acid (cmo 5 U), uracil 5-oxyacetate (mcmo 5 U), 5-carboxymethyl-uracil (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm 5 U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uracil (mcm 5U), 5-methoxycarbonylmethyl-2-thiouracil (mcm 5 s 2 U), 5-aminomethyl-2-thiouracil (nm 5 s 2 U), 5-methylaminomethyluracil (mnm 5 U), 5-methylaminomethyl-2-thiouracil (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouracil (mnm 5 se 2 U), 5-carbamoylmethyluracil (ncm 5 U), 5-carboxymethylaminomethyl-uracil (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm 5 s 2 U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurine methyl-uracil (τm 5 U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uracil (τm 5 5s 2 U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uracil (m 5 U, i.e., with the nucleobase deoxythymidine), 1-methyl-pseudouridine (m 1 ψ), 1-ethyl-pseudouridine (Et 1 ψ), 5-methyl-2-thiouracil (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m 5 D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl) uracil (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uracil (m 5U), 5-(isopentenylaminomethyl)-2-thio-uracil (m 5 s 2 U), 5,2'-O-dimethyl-uridine (m 5 Um), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil and 5-[3-(1-E-propenylamino)]uracil.

[0151] In specific embodiments, nucleic acid molecules of the present disclosure comprise modifications to uracil. In specific embodiments, nucleic acid molecules of the present disclosure comprise one or more pseudouracils (ψ). In specific embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the uracils in the nucleic acid molecules of the present disclosure are replaced by pseudouracils (ψ). In specific embodiments, all (100%) uracils in the nucleic acid molecules of the present disclosure are replaced by pseudouracils (ψ).

[0152] In specific embodiments, the nucleic acid molecules of the present disclosure comprise one or more 1-methylpseudouracils (m1ψ). In specific embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the uracils in the nucleic acid molecules of the present disclosure are substituted with 1-methyl-pseudouracils (m1ψ). In specific embodiments, all (100%) of the uracils in the nucleic acid molecules of the present disclosure are substituted with 1-methylpseudouracils (m1ψ).

[0153] Therapeutic nucleic acid molecules as described herein can be isolated or synthesized using methods known in the art. In some embodiments, the DNA or RNA molecules used in conjunction with the present disclosure are chemically synthesized. In other embodiments, the DNA or RNA molecules used in conjunction with the present disclosure are isolated from natural sources.

[0154] In some embodiments, the mRNA molecules used in conjunction with the present disclosure are biosynthesized using host cells. In specific embodiments, mRNA is produced by transcribing the corresponding DNA using host cells. In some embodiments, the DNA sequence encoding the mRNA sequence is integrated into an expression vector using methods known in the art, and the vector is then introduced into a host cell (e.g., E. coli). The host cell is then cultured under suitable conditions to produce mRNA transcripts. Other methods for producing mRNA molecules from DNA are known in the art. For example, in some embodiments, a cell-free (in vitro) transcription system comprising an enzyme of the host cell's transcriptional machinery can be used to produce mRNA transcripts. An exemplary cell-free transcription reaction system is described in Example 4 of the present invention.

[0155] 3.6 Methods for preparing mRNA

[0156] The present invention also provides a method for preparing mRNA containing an open reading frame (ORF) encoding urate oxidase, a functional fragment or variant thereof, comprising the following steps: 1) synthesizing a DNA fragment for transcribing the mRNA, and cloning the DNA fragment into an expression vector to obtain a recombinant plasmid; 2) transferring the recombinant plasmid into a host cell, amplifying the plasmid, extracting the plasmid, and digesting the resulting plasmid with a restriction endonuclease to obtain linearized DNA for in vitro transcription of mRNA; and 3) transcribing the linearized DNA in vitro to obtain the target mRNA.

[0157] In some embodiments, a DNA fragment is synthesized to transcribe an mRNA containing an open reading frame (ORF) encoding uricase, a functional fragment thereof, or a variant thereof, and the DNA fragment is cloned into an expression plasmid to obtain a recombinant plasmid. In some embodiments, in addition to containing a DNA fragment corresponding to the mRNA encoding uricase, the DNA fragment should also contain a T7 promoter sequence at the 5' end and a specific restriction endonuclease recognition sequence at the 3' end, wherein the restriction endonuclease can be selected from BspQI, BsaI, BsmI, NotI, etc. The present invention does not specifically limit the method for synthesizing the DNA fragment corresponding to the above-mentioned mRNA, and conventional DNA synthesis methods in the art can be used. In a preferred embodiment, the DNA fragment is synthesized by a commercial biotechnology company. In some embodiments, the expression plasmid does not contain a T7 promoter sequence and the recognition sequence of the specific restriction endonuclease (restriction endonuclease can be selected from BspQI, BsaI, BsmI, NotI, etc.) used above. In some embodiments, the expression plasmid is preferably a pUC-GW plasmid. The plasmid map of the pUC-GW plasmid is shown in Figure 2. In a preferred embodiment, the DNA fragment is cloned into an expression plasmid by enzyme digestion. The present invention has no particular limitation on the specific operations of enzyme digestion and ligation, and conventional enzyme digestion and ligation operations in the art can be used.

[0158] After obtaining the recombinant plasmid, the recombinant plasmid is introduced into the host cell, amplified, the plasmid is extracted, and the obtained plasmid is digested with a restriction endonuclease to obtain a linearized DNA fragment for in vitro expression of mRNA. In a preferred embodiment, the host cell is an Escherichia coli cell or a competent form thereof. The present invention does not specifically limit the method of introduction, and conventional introduction methods in the art can be used. After obtaining the recombinant cells, positive recombinant cells are screened and colony sequencing is performed. In one embodiment, the screening of the positive recombinant cells is carried out on a solid culture medium containing kanamycin (kan), followed by colony PCR to select kan-resistant colonies. In some embodiments, the amplification procedure of the colony PCR is as follows: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 2 minutes, 34 cycles; and final extension at 72°C for 10 minutes. After the PCR amplification is completed, the colonies of the target bands are preferably determined by agarose gel electrophoresis, and then sequenced for verification. In some embodiments, the parameters of the agarose gel electrophoresis detection are as follows: 1% agarose, 5V / cm, 40min. In some embodiments, the plasmid of the recombinant cell with correct sequencing is extracted. The present invention does not specifically limit the extraction method of the plasmid, and preferably a plasmid extraction kit is used. In some embodiments, the specific restriction endonuclease mentioned above (the restriction endonuclease can be selected from BspQI, BsaI, BsmI, NotI, etc.) is used for enzyme digestion to obtain linearized DNA fragments of in vitro expressed mRNA. In some embodiments, the enzyme digestion system is designed with 50μl, preferably as follows:

[0159] In some embodiments, after the enzyme cleavage reaction is completed, the amplified product is preferably subjected to agarose gel electrophoresis to determine whether the reaction is complete. The agarose gel electrophoresis detection parameters are preferably as follows: 1% agarose, 5V / cm, 40min. In some embodiments, the reaction is considered complete when only one band appears on agarose gel electrophoresis. After the enzyme cleavage reaction is completed, the linearized DNA fragment is preferably purified. There is no special limitation on the purification method, and conventional purification methods in the art can be used. In the specific implementation process of the present invention, it is preferably carried out using a DNA purification kit. After the purification, NanoDrop is preferably used to detect the concentration of the purified linearized DNA template, as well as the OD 260nm / OD 280nm , OD 260nm / OD 230nm The ratio of OD 260nm / OD 280nm When the value is between 1.6 and 1.8, the linearized template is considered qualified.

[0160] After obtaining the linearized DNA template, the linearized DNA template is subjected to in vitro transcription to obtain the mRNA. The mRNA in vitro transcription system is designed as a 20 μl system and includes the following components:

[0161] In some embodiments, the Enzyme Mix includes T7 RNA polymerase, an RNase inhibitor, and an inorganic pyrophosphatase. In some embodiments, the in vitro RNA synthesis reaction is preferably performed in a 200 μl RNase-free tube at 37°C for 2 hours. The reaction reagents in the RNA in vitro transcription system are added in the following order: water, nucleotides, cap analog, transcription buffer, Enzyme Mix, and linearized DNA template.

[0162] After the in vitro transcription of the RNA is completed, it is preferably verified by agarose gel electrophoresis whether the in vitro synthesis of the mRNA is successful. The detection parameters of the agarose gel electrophoresis are as follows: 1% agarose, 5V / cm, 10min. In some embodiments, the appearance of the expected target band by agarose gel electrophoresis indicates that the reaction is successful. In some embodiments, the mRNA synthesized in vitro is subjected to steps such as removing the DNA template and recovering and purifying the mRNA. In some embodiments, the removal of the DNA template is preferably achieved by DNase I digestion. In some embodiments, the digestion is carried out as follows: 2μl of DNase I is mixed with the solution after the RNA in vitro transcription reaction, and incubated at 37°C for 15min. After the digestion is completed, it is preferably performed to detect residual DNA fragments. In some embodiments, there is no limitation on the method for recovering and purifying the mRNA, and conventional purification methods in the art can be used. In the specific implementation process of the present invention, it is preferably carried out using an RNA purification kit. After recovering and purifying the mRNA, the mRNA quality test is performed, and the quality test includes the concentration of the mRNA, the OD value of the mRNA, and the mRNA concentration. 260nm / OD 280nm ,OD 260nm / OD 230nm The ratio of OD 260nm / OD 280nm The range is 1.8-2.1 and the OD 260nm / OD 230nm When the range is greater than 2.0, the mRNA is considered qualified. After the mRNA is recovered and purified, the purified mRNA is preferably packaged.

[0163] 3.7 Lipid Nanoparticles (LNP)

[0164] In some embodiments, the therapeutic composition may also include a delivery vehicle. The mRNA described herein may be formulated in nanoparticles or other delivery vehicles to prevent the mRNA from being degraded when delivered to a subject. In some embodiments, the mRNA may be encapsulated in nanoparticles. In a specific embodiment, nanoparticles are particles having at least one size (e.g., diameter) less than or equal to 1000nm, less than or equal to 500nm, or less than or equal to 200nm. In a specific embodiment, nanoparticles include lipids. Lipid nanoparticles may include, but are not limited to, liposomes and micelles. In a specific embodiment, the lipid nanoparticles may include cations and / or ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, pegylated lipids and / or structural lipids, or a combination thereof. In certain embodiments, lipid nanoparticles include one or more mRNA described herein, for example, mRNA encoding a protein of interest (e.g., uricase).

[0165] The delivery vehicle in the compositions described herein can be nano lipid particles. The nano lipid particles can comprise one or more cations and / or ionizable lipids." cationic lipids" generally refer to lipids that carry any number of net positive charges at a certain pH (such as physiological pH). The cationic lipids may include, but are not limited to, 3-(didodecylamino)-N1, N1, 4-triadecyl-1-piperazineethylamine (KL10), N1-[2-(docosylamino)ethyl]-N1, N4, N4-triadecyl-1, 4-piperazinediethylamine (KL22), 14,25-tricosyl-15,18,21,24-tetraazaoctapore and alkane (KL25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, Octyl-CLinDMA, octyl-CLinDMA (2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Chol, DOSPA, DOGS, DODAP, DODMA and DMRIE. In addition, many commercial cationic and / or ionizable lipids may be used, for example, (including DOTMA and DOPE) and (including DOSPA and DOPE). For example, the cationic lipid can be DLin-MC3-DMA. In some embodiments, the molar ratio of the cationic lipid in the lipid nanoparticle is about 40-70%. In a specific embodiment, the molar ratio of the cationic lipid in the lipid nanoparticle is about 50%.

[0166] In some embodiments, the nano lipid particles may comprise one or more non-cationic lipids. The non-cationic lipids may include anionic lipids. Anionic lipids suitable for use in the lipid nanoparticles of the present invention may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylphosphatidylethanolamine, and other neutral lipids to which anionic groups are attached.

[0167] In some embodiments, the non-cationic lipids may include neutral lipids having a net charge of zero at physiological pH. Neutral lipids suitable for use in lipid nanoparticles of the present invention may include phospholipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DP ... Oleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or a mixture thereof. In addition, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. For example, the neutral lipids described herein can be selected from DOPE, DSPC, DPPC, POPC or any related phosphatidylcholine. In a specific embodiment, the neutral lipid is DSPC. In some embodiments, the molar ratio of the neutral lipid in the lipid nanoparticle is about 5-15%. In a specific embodiment, the molar ratio of the neutral lipid in the lipid nanoparticle is about 10%.

[0168] In some embodiments, the nano lipid particle can include lipid conjugates, and lipid conjugates include lipid moiety and polymer moiety, for example, the PEGylated lipid including lipid moiety and polyethylene glycol (PEG) moiety. Suitable lipid conjugates for use in the present invention include dimyristoylphosphatidylethanolamine-poly (ethylene glycol) 2000 (DMPE-PEG2000), DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-c-DOMG, PEG2000-c-DOPG etc. The molecular weight of operable poly (ethylene glycol) can be in the range of about 500 to about 10,000Da, or about 1,000 to about 5,000Da. The addition of these components can prevent lipid aggregation, and can also increase circulation duration, and is easy to lipid-nucleic acid composition delivery to target cells, or quickly releases nucleic acid. In a specific embodiment, the nano lipid particle can include PEG2000-DMG. In some embodiments, the molar ratio of the polyethylene glycol (PEG)-modified lipid molecules in the lipid nanoparticles is about 0.5-2%. In a specific embodiment, the molar ratio of the polyethylene glycol (PEG)-modified lipid molecules in the lipid nanoparticles is about 1.5%.

[0169] In some embodiments, the nanolipid particles may further comprise cholesterol. In some embodiments, the molar ratio of the cholesterol in the lipid nanoparticles is about 30-45%. In a specific embodiment, the molar ratio of the cholesterol in the lipid nanoparticles is about 38.5%.

[0170] In some embodiments, the nanolipid particles may include cationic lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipid molecules. In some embodiments, the molar ratio of the cationic lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipid molecules may be 45-55:35-45:5-15:0.5-2. In certain embodiments, the molar ratio of the cationic lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipid molecules may be 50:38.5:10:1.5.

[0171] 3.8 Nanoparticle Composition

[0172] On the one hand, nucleic acid molecules as described herein are formulated for in vitro and in vivo delivery. In particular, in some embodiments, nucleic acid molecules are formulated into lipid-containing compositions. In some embodiments, lipid-containing compositions form lipid nanoparticles that enclose nucleic acid molecules in lipid shells. In some embodiments, lipid shells protect nucleic acid molecules from degradation. In some embodiments, lipid nanoparticles also help to transport enclosed nucleic acid molecules to intracellular compartments and / or mechanisms to provide expected preventive effects. In certain embodiments, when present in lipid nanoparticles, nucleic acids can resist degradation by nucleases in aqueous solutions. Lipid nanoparticles comprising nucleic acids and methods for their preparation are known in the art, such as those disclosed in U.S. Patent Publication No. 2004 / 0142025, U.S. Patent Publication No. 2007 / 0042031, PCT Publication No. WO 2017 / 004143, PCT Publication No. WO 2015 / 199952, PCT Publication No. WO 2013 / 016058, and PCT Publication No. WO 2013 / 086373, the entire disclosures of which are incorporated herein by reference in their entirety.

[0173] For example, multilamellar vesicles (MLVs) can be prepared by existing techniques, for example, by depositing a selected lipid on the inner wall of a suitable container or vessel; by dissolving the lipid in a suitable solvent and then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying. An aqueous phase can be added to a rotating vessel, which results in the formation of MLVs. Unilamellar vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0174] Nanoparticle compositions as used herein include delivery vehicles (e.g., nanolipid particles), wherein mRNA can be associated with the surface of the lipid delivery vehicle and encapsulated therein. For example, when preparing nanoparticle compositions of the present disclosure, the cationic lipid delivery vehicle can be associated with mRNA through electrostatic interaction.

[0175] In the present invention, consider the size of target cell or tissue and the liposome application degree to be prepared to select the appropriate size of described lipid delivery vector (for example, lipid nano particle).In a specific embodiment, mRNA can be delivered to specific cell or tissue.For example, for targeting hepatocyte, can determine the size of lipid delivery vector (for example, lipid nano particle), make its size ratio the fenestration of endothelial lining liver sinusoidal space in liver less, make lipid delivery vector (for example, lipid nano particle) can easily permeate these endothelial fenestrations to arrive target hepatocyte.Lipid delivery vector (for example, lipid nano particle) can have enough large diameter to limit or obviously avoid being distributed in some cell or tissue.In some embodiments, the size (for example, diameter) of described lipid delivery vector (for example, lipid nano particle) can be in the range of about 25 to 250nm, for example, less than about 250nm, 175nm, 150nm, 125nm, 100nm, 75nm, 50nm, 25nm or 10nm. For example, the size (e.g., diameter) of the lipid delivery vehicle (e.g., lipid nanoparticle) can be in the range of about 25 to 250 nm, e.g., about 50 to 200 nm, about 75 to 175 nm, about 75 to 150 nm, or about 75 to 125 nm.

[0176] Nanoparticle compositions that can be used in conjunction with the present disclosure include, for example, lipid nanoparticles (LNPs), nanolipoprotein particles, liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayers can include one or more ligands, proteins, or channels.

[0177] 3.9 Preparation

[0178] According to the present disclosure, the nanoparticle compositions described herein can include at least one lipid component and one or more additional components, such as therapeutic and / or prophylactic agents. Nanoparticle compositions can be designed for one or more specific applications or goals. The components of a nanoparticle composition can be selected based on the specific application or goal, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, a specific formulation of a nanoparticle composition can be selected for a specific application or goal based on, for example, the efficacy and toxicity of a specific combination of each component.

[0179] The lipid component of the nanoparticle composition can include ionizable lipids, phospholipids (e.g., unsaturated lipids such as DOPE or DSPC), PEG lipids, and structural lipids. The components of the lipid component can be provided in a specific ratio.

[0180] In one embodiment, a nanoparticle composition is provided herein comprising a cationic or ionizable lipid compound, a therapeutic agent as provided herein, and one or more excipients. In one embodiment, the one or more excipients are selected from neutral lipids, phospholipids, steroids, and polymer-conjugated lipids. In one embodiment, the therapeutic agent is encapsulated within or associated with the lipid nanoparticles.

[0181] Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed for delivering therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof in mammals. The physicochemical properties of nanoparticle compositions can be altered to increase selectivity for specific body targets. For example, particle size can be adjusted based on the window size of different organs. The therapeutic and / or prophylactic agents contained in the nanoparticle compositions can also be selected based on one or more desired delivery targets. For example, therapeutic and / or prophylactic agents can be selected for specific indications, conditions, diseases, or disorders and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery). In certain embodiments, nanoparticle compositions can include mRNA encoding a polypeptide of interest, which can be translated intracellularly to produce a polypeptide of interest. Such compositions can be designed to be specifically delivered to a specific organ. In certain embodiments, compositions can be designed to be specifically delivered to the liver of a mammal.

[0182] The amount of the therapeutic and / or prophylactic agent in the nanoparticle composition can depend on the size, composition, desired target and / or application, or other characteristics of the nanoparticle composition, as well as the characteristics of the therapeutic and / or prophylactic agent. For example, the amount of RNA that can be used in the nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of the therapeutic and / or prophylactic agent and other components (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the wt / wt ratio of the lipid component to the therapeutic and / or prophylactic agent in the nanoparticle composition can be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt / wt ratio of the lipid component to the therapeutic and / or prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 14: 1. In certain embodiments, the wt / wt ratio is about 20: 1. In certain embodiments, the wt / wt ratio is about 40: 1. The amount of the therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0183] In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in the one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N:P ratio is selected. The one or more RNAs, lipids, and amounts thereof may be selected to provide an N:P ratio of about 2:1 to about 30:1, e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio may be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio can be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N:P ratio can be about 5.67:1.

[0184] The physical properties of a nanoparticle composition can depend on its components. For example, a nanoparticle composition comprising cholesterol as a structural lipid can have different characteristics than a nanoparticle composition comprising a different structural lipid. Similarly, the characteristics of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher molar ratio of phospholipids can have different characteristics than a nanoparticle composition comprising a lower molar ratio of phospholipids. Characteristics can also vary depending on the method and conditions used to prepare the nanoparticle composition.

[0185] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle compositions. Zeta potential can be measured using dynamic light scattering or potentiometric methods (e.g., potentiometric titration). Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malverm Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of the nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0186] In various embodiments, the average size of the nanoparticle composition can be between tens of nanometers and hundreds of nanometers. For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the nanoparticle composition can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the average size of the nanoparticle composition can be from about 70 nm to about 100 nm. In some embodiments, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0187] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A smaller polydispersity index (e.g., less than 0.3) generally indicates a narrower particle size distribution. The polydispersity index of the nanoparticle composition can be from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be from about 0.05 to about 0.15.

[0188] The zeta potential of a nanoparticle composition can be used to indicate the zeta potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low positive or negative charges are generally desirable because highly charged species can interact undesirably with cells, tissues, and other components in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0189] The encapsulation efficiency of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent encapsulated or otherwise associated with the nanoparticle composition after preparation relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the nanoparticle composition before and after disrupting the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0190] The nanoparticle compositions may optionally include one or more coatings. For example, the nanoparticle compositions may be formulated into capsules, films, or tablets having a coating. The capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0191] 3.10 Pharmaceutical Compositions

[0192] According to the present disclosure, nanoparticle compositions can be formulated, in whole or in part, into pharmaceutical compositions. A pharmaceutical composition can comprise one or more nanoparticle compositions. For example, a pharmaceutical composition can comprise one or more nanoparticle compositions comprising one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition can further comprise one or more pharmaceutically acceptable excipients or adjuvants, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents can be found, for example, in Remington's The Science and Practice of Pharmacy, 21st ed., A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and adjuvants can be used in any pharmaceutical composition, unless any conventional excipient or adjuvant is incompatible with one or more components of the nanoparticle composition. An excipient or adjuvant is incompatible with a component of the nanoparticle composition if the combination of the excipient or adjuvant with the component of the nanoparticle composition would result in any undesirable biological or other deleterious effect.

[0193] In some embodiments, the one or more excipients or adjuvants may constitute more than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, the one or more excipients or adjuvants may constitute 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical customary percentage. In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and / or the International Pharmacopoeia.

[0194] The relative amounts of the one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated and further on the route of administration of the composition. For example, a pharmaceutical composition may contain between 0.1% and 100% (wt / wt) of the one or more nanoparticle compositions.

[0195] In some embodiments, nanoparticle compositions and / or pharmaceutical compositions of the disclosure are stored and / or shipped refrigerated or frozen (e.g., stored at 4°C or lower, for example, between about -150°C and about 0°C, or between about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C)). For example, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein are stable for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months at a temperature of, for example, 4° C. or less (e.g., between about 4° C. and −20° C.). In one embodiment, the formulation is stable for at least 4 weeks at about 4° C. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of: Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pH of the pharmaceutical compositions of the present disclosure is between about 7 and 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5 and 8, or between 7 and 7.8). For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5-8, which is suitable for storage and / or transport at, for example, about -20°C. For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and PBS, and has a pH of about 7-7.8, which is suitable for storage and / or transport at, for example, about 4°C or lower. In the context of the present disclosure, "stability," "stabilized," and "stable" refer to the resistance of the nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size changes, aggregation, changes in encapsulation, etc.) under given manufacturing, preparation, transportation, storage, and / or use conditions, for example, when stress is applied, such as shear force, freeze / thaw stress, etc.

[0196] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including patients or subjects who may benefit from the therapeutic effects provided by delivering a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof, such as the renal system. Although the descriptions of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein are primarily directed to compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Modifications to compositions suitable for administration to humans in order to make the compositions suitable for administration to various animals are well known, and such modifications can be designed and / or performed by a veterinary pharmacologist of ordinary skill with only ordinary experimentation (if any). It is contemplated that subjects to whom the compositions may be administered include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0197] Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known or later developed in the art of pharmacology. Generally, such preparation methods comprise combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if desired or necessary, dividing, shaping, and / or packaging the product into desired single-dose or multi-dose units.

[0198] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or a convenient fraction of such a dose, such as half or one-third of such a dose.

[0199] Pharmaceutical compositions can be prepared into various forms suitable for various routes and methods of administration. For example, pharmaceutical compositions can be prepared into liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0200] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and / or elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may also contain additional therapeutic and / or prophylactic agents, additional agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and / or flavoring agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizer, such as Cremophor TM , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and / or combinations thereof.

[0201] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersants, wetting agents and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions and / or emulsions in non-toxic parenteral acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP and isotonic sodium chloride solution. Sterile fixed oils are typically used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injections.

[0202] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0203] 3.11 Treatment Methods

[0204] The present disclosure features methods of delivering therapeutic and / or prophylactic agents to mammalian cells or organs, producing proteins of interest in mammalian cells, and treating a disease or condition in a mammal in need thereof, the methods comprising administering to the mammal a nanoparticle composition comprising the therapeutic and / or prophylactic agent and / or contacting mammalian cells with the nanoparticle composition.

[0205] In one aspect, the present disclosure provides a method for reducing uric acid levels in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a therapeutic nucleic acid as described herein or a pharmaceutical composition comprising the therapeutic nucleic acid. In some embodiments, the subject is a human or non-human mammal.

[0206] In some embodiments, the nucleic acid or pharmaceutical composition is administered intramuscularly, subcutaneously, or intravenously.

[0207] In some embodiments, the nucleic acid-encapsulated lipid nanoparticles are endocytosed by cells in the subject.

[0208] In some embodiments, the nucleic acid is expressed by cells in the subject.

[0209] In a further embodiment, the administration is intravenous.

[0210] In further embodiments, the administration is one or more times at a frequency of about once a day, about once every two days, about once every three days, about once every four days, about once every five days, about once every six days, about once a week, about once every two weeks, or about once a month.

[0211] In another aspect, the present disclosure provides a method for treating or preventing gout, gouty arthritis, hyperuricemia or tumor lysis syndrome in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the therapeutic nucleic acid described herein or a pharmaceutical composition comprising the therapeutic nucleic acid.

[0212] In some embodiments, the subject is a human or non-human mammal.

[0213] In some embodiments, the nucleic acid or pharmaceutical composition is administered intralesionally, subcutaneously, or intravenously.

[0214] In some embodiments, the nucleic acid-encapsulated lipid nanoparticles are endocytosed by cells in the subject.

[0215] In some embodiments, the nucleic acid is expressed by cells in the subject.

[0216] In a further embodiment, the administration is intravenous.

[0217] In further embodiments, the administration is one or more times at a frequency of about once a day, about once every two days, about once every three days, about once every four days, about once every five days, about once every six days, about once a week, about once every two weeks, or about once a month.

[0218] 4. Technical Effects of the Present Disclosure

[0219] Through creative work, the inventors discovered that by delivering the nucleic acid therapeutic agent described herein into the body and utilizing human cells to express urate oxidase, the metabolism of uric acid in the blood and / or liver can be accelerated, significantly reducing blood uric acid levels, thereby treating gout, gouty arthritis, hyperuricemia or tumor lysis syndrome.

[0220] The therapeutic uricase nucleic acids disclosed herein have the following advantages:

[0221] 1. mRNA expression in host cells is highly persistent, significantly reducing dosing frequency without the need for exogenous molecules such as PEG;

[0222] 2. By screening elements such as ORF, 5'-UTR, 3'-UTR, and signal peptide, we found combinations with optimal expression levels and different secretion ratios;

[0223] 3. Reduce the immunogenicity of mRNA and improve in vivo stability by introducing non-natural nucleotides;

[0224] 4. Liposome technology is used to achieve liver-targeted drug delivery, specifically expressing uricase in the liver, the tissue where uric acid is initially produced and where uricase naturally exists, and screening is performed based on the secretion ratio of the generated uricase. The present invention screened three types of uricase oxidase expression mRNA with different properties: ① highly secreted uricase oxidase expression mRNA (secretion ratio > 90%); ② highly cell-retained uricase oxidase expression mRNA (secretion ratio < 30%); and ③ uricase oxidase expression mRNA with a moderate secretion level (secretion ratio > 30%, but less than 80%). Among them, highly secreted uricase oxidase expression mRNA and moderate secretion level uricase expression mRNA have unique advantages in different indications, as follows:

[0225] A. Highly secreted uricase mRNA expression (secretion ratio >90%): For patients with tumor lysis syndrome (TLS), rapid clearance of circulating uric acid is the primary goal of treatment, and patients do not require long-term dosing. Existing therapies require five consecutive days of intravenous infusion of rasburicase. The highly secreted uricase mRNA expression (secretion ratio >90%) in this study not only rapidly clears uric acid from the peripheral circulation, but also significantly reduces dosing frequency due to the persistence of mRNA expression.

[0226] B. Moderate secretion level of urate oxidase mRNA expression (secretion ratio >30%, but less than 80%): For gout patients with more severe gout, moderate secretion level of urate oxidase mRNA expression (secretion ratio >30%, but less than 80%) can not only effectively control the patient's uric acid level and reduce the humoral immune response, but also ensure that a certain proportion of urate oxidase is secreted into the peripheral circulation, thereby promoting the dissolution of gout in the joints.

[0227] 5. By introducing a spacer sequence, the expression of urate oxidase protein produced by the translation of highly secreted urate oxidase mRNA in cells was significantly increased;

[0228] 6. By introducing a half-life extension element, a longer-acting uricase mRNA therapeutic composition is obtained, thereby significantly increasing the expression of uricase protein in the body.

[0229] Sequence Listing

[0230] Example

[0231] The technical features involved in the different implementation plans described in this article can be implemented in combination with each other.

[0232] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0233] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0234] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are well described in the literature, for example, by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc. The reagents and raw materials used in the present invention are all commercially available.

[0235] Example 1. Design of Arthritis Bacterium Urate Oxidase mRNA

[0236] [Corrected 28.08.2024 in accordance with Rule 26] To identify the most suitable functional elements for expressing uricase or its fusion protein in human cells as mRNA sequences, with varying secretion efficiencies, the inventors first screened for functional elements using Arthrobacterium uricase (SEQ ID NO:5) as an example. The inventors introduced a signal peptide sequence (SEQ ID NO:22) into the N-terminus of the amino acid sequence of Arthrobacterium uricase (SEQ ID NO:5) to form a uricase fusion protein. The inventors then codon-optimized the coding sequence of this fusion protein to design an ORF sequence (SEQ ID NO:67). In addition, the inventors used four 5'-UTR sequences (SEQ ID NOs: 54-57) and five 3'-UTR sequences (SEQ ID NOs: 58-62) as candidate functional elements. Following the structure of 5' cap-5'-UTR coding sequence-codon-optimized nucleotide sequence encoding uricase fusion protein-3'-UTR coding sequence-3'-polyadenylation (as shown in Figure 1), these codon-optimized nucleotide sequences were combined with various other functional elements to assemble mRNAs (SEQ ID NOs: 213-235). For each sequence, two mRNAs were prepared: one encoding a uricase variant without a tag and the other encoding a uricase variant with a Flag tag at the C-terminus for subsequent expression verification.

[0237] Example 2. Preparation of linearized DNA template

[0238] 2.1. Preparation of recombinant plasmid

[0239] 1) Synthesize DNA fragments corresponding to SEQ NO IDs: 213-380, 390-393 (with and / or without Flag tags). The DNA fragments should contain a T7 promoter sequence at the 5' end, DNA corresponding to the mRNA encoding the uricase fusion protein, and a BspQI or NotI recognition sequence at the 3' end.

[0240] 2) The synthesized target DNA fragment and pUC-GW plasmid (as shown in Figure 2) were digested with enzymes respectively.

[0241] 3) Use T4 ligase (Thermo Scientific, EL0011) to perform in vitro ligation to obtain the recombinant plasmid.

[0242] 4) The above recombinant plasmid was transformed into competent Escherichia coli and + Culture on solid medium for 12 h.

[0243] 5) Select single colonies for colony PCR and select colonies containing the target band for sequencing.

[0244] 6) The correctly sequenced colonies were expanded and cultured before plasmid extraction.

[0245] 2.2 Preparation of linearized DNA

[0246] 1) After obtaining the recombinant plasmid, use BsqQI or NotI (purchased from NEB, R0712 / R3189) to digest the plasmid to obtain linearized DNA for in vitro mRNA expression.

[0247] 2) After the digestion reaction is complete, aspirate 1 μl of the reaction solution and perform DNA agarose gel electrophoresis to confirm the reaction is complete. The agarose gel electrophoresis parameters are as follows: 1% agarose (purchased from Biowest, 111860), 5 V / cm, 40 min. When a single band is detected by electrophoresis and the size is consistent with the expected value, the digestion reaction is considered complete and the linearized DNA template has been successfully prepared.

[0248] 3) Purify the linearized DNA using the Thermo Scientific K0701 GeneJET PCR Purification Kit.

[0249] 4) Use NanoDrop to detect the concentration of the purified linearized DNA template and OD 260nm / OD 280nm ,OD 260nm / OD 230nm When OD 260nm / OD 280nm Between 1.8 and 2.0, OD 260nm / OD 230nm Between 1.6 and 2.2 is considered qualified.

[0250] Experimental results: All linearized DNA templates were detected by agarose gel electrophoresis as a single band with the expected size, OD 260nm / OD 280nm All between 1.8 and 2.0, OD 260nm / OD 230nm All were between 1.6 and 2.2, meeting the requirements for in vitro transcription.

[0251] Example 3. In vitro transcription

[0252] 3.1. Obtain purified linearized DNA and perform in vitro transcription in a thermostatic reactor. The in vitro transcription reaction system is as follows:

[0253] The NTPs include ATP, UTP, GTP, and CTP. Any of these NTPs can be replaced by corresponding modified nucleotides. The in vitro transcription procedure is 37°C for 2 hours. In addition, the Cap analogue mentioned above refers to the Cap1 analogue, whose structure is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG. All the above raw materials were purchased from Zhaowei Biotechnology.

[0254] After the in vitro transcription reaction is complete, aspirate 1 μl of the reaction solution and perform agarose gel electrophoresis to confirm the success of the reaction. The parameters for agarose gel electrophoresis are as follows: 1% agarose (purchased from Biowest, 111860), 5 V / cm, 10 min. If a band of the expected size appears during electrophoresis, the reaction is considered successful. If a band the size of the DNA template still appears during electrophoresis, residual DNA is present and DNase digestion is required to remove the DNA.

[0255] 3.3. Add 2 μl of DNase I (purchased from NEB, M0303) to the mRNA in vitro transcription reaction solution and digest at 37°C for 15 minutes.

[0256] After the digestion reaction is complete, aspirate 1 μl of the reaction solution and perform agarose gel electrophoresis to confirm the success of the reaction. The parameters for agarose gel electrophoresis are as follows: 1% agarose (purchased from Biowest, 111860), 5 V / cm, 10 min. The reaction is considered successful when a band corresponding to the expected mRNA size appears and the band corresponding to the DNA template size disappears.

[0257] 3.5. Use NEB T2050 Monarch RNA Cleanup Kit to recover and purify RNA from the reaction solution after DNase I digestion.

[0258] 3.6. Pipette 1 μl of purified mRNA and perform agarose gel electrophoresis using the following parameters: 1% agarose (purchased from Biowest, 111860), 5 V / cm, 10 min. The mRNA preparation is considered qualified when a band of the expected size appears on the electrophoresis, and the band is clear and has no tailing.

[0259] 3.7. Use NanoDrop to detect the concentration of recovered purified mRNA and OD 260nm / OD 280nm , OD 260nm / OD 230nm The ratio of OD 260nm / OD 280nm Range is 2.0-2.2, OD 260nm / OD 230nmWhen the mRNA is within the range of 1.8-2.2, the prepared mRNA is considered qualified. The prepared mRNA is then immediately aliquoted and frozen at -80°C.

[0260] Example 4 Screening of modified nucleotides

[0261] 4.1. mRNA Preparation

[0262] Taking SEQ ID NO: 213 as an example, the in vitro transcription method in Example 3 was used to synthesize SEQ ID NO: 213 mRNA sequences with different modified nucleotides to screen for modified nucleotides that are most suitable for the expression of urate oxidase mRNA in human cells. The modified nucleotides used in this example include 5-methylcytidine, 5-methoxyuridine, pseudouridine, N6-methyladenosine, and N1-methylpseudouridine (all purchased from Zhaowei Bio). After in vitro transcription to prepare mRNA, the mRNA was purified and quality tested according to the method described in Example 3.

[0263] Results: The yield of mRNA modified with N6-methylpseudouridine was less than 2 μg, and no band was observed on agarose gel. Agarose gel electrophoresis of all other modified mRNAs showed a band of the expected size, which was clear and had no tailing; OD 260nm / OD 280nm All between 2.0 and 2.2, OD 260nm / OD 230nm All are between 1.8 and 2.2.

[0264] The above results show that, except for the N6-methylpseudouridine-modified mRNA, which failed to be prepared, all other modified mRNAs were prepared qualified and can be used for the subsequent expression of urate oxidase fusion protein.

[0265] Cell transfection

[0266] 1) Cell Culture: In this example, HEK293T cells purchased from ATCC were transfected with modified mRNA to detect urate oxidase expression. HEK293T cells were cultured in DMEM (Gibco, 11965092) supplemented with 10% fetal bovine serum (Gibco, 10099).

[0267] 2) Cell inoculation: HEK293T cells in good growth condition were taken, digested, resuspended, counted, and diluted to 5*10 5 / ml, mix well and inoculate 1ml / well of cell suspension into a 12-well plate.

[0268] 3) After inoculation, the cells were cultured for approximately 24 hours, the cell status in the 12-well plate was observed, and cell transfection was performed when the cell confluence reached 70-90%. Cell transfection was performed using Lipofectamine RNAiMAX (purchased from Invitrogen, 13778500).

[0269] 4) Prepare transfection reagent: Mix 50 μl of Opti-MEM (Gibco, 31985070) with 3 μl of Lipofectamine RNAiMAX. Also, mix 50 μl of Opti-MEM with 2 μg of prepared mRNA. Then, mix the diluted mRNA with the diluted RNAiMAX and incubate at room temperature for 5 minutes.

[0270] 5) Aspirate the cell supernatant from the 12-well plate, wash once with PBS, add the transfection reagent to the cells, and fill the volume to 1 ml with culture medium. Harvest the cells and / or cell supernatant 24-48 hours after transfection.

[0271] 4.3 Protein expression Western blot detection

[0272] By detecting the expression of the Flag tag introduced at the C-terminus of the coding sequence, the expression level of the urate oxidase protein containing the above-mentioned modified nucleotide mRNA is determined, and the preferred modified nucleotide is screened. The protein expression detection steps are as follows:

[0273] 1) Cell Lysis: Cells in a 12-well plate were digested with trypsin, then resuspended in complete medium, centrifuged at 1000 rpm for 5 minutes, washed once with PBS, and centrifuged at 1000 rpm for 5 minutes. 100 μl of RIPA (Thermo Scientific, 89901) and 1 μl of protease inhibitor (Thermo Scientific, 78442) were added to each well of cells. The cells were incubated on ice for 30 minutes and centrifuged at 12,000 g for 15 minutes at 4°C. The supernatant was aspirated and the protein concentration was determined using a BCA kit (Beyotime, P0010).

[0274] 2) Western blotting: Add 5× SDS loading buffer (purchased from Beyotime, P0015) to the cell lysate sample and incubate at 100°C for 5 minutes before loading the sample. Load 1 μg of cell lysate sample or 10 μl of supernatant sample per well. Electrophoresis was performed at 70 V for 30 minutes or 110 V for 90 minutes.

[0275] 3) Transfer: Use GenScript's rapid membrane transfer instrument to transfer the proteins on the SDS gel to a 0.45 μm pore size PVDF membrane.

[0276] 4) Blocking: The membrane was blocked with Western blocking buffer (purchased from Beyotime, P0023B) at room temperature for 2 hours, and then washed with TBST three times for 5 minutes each time.

[0277] 5) Primary antibody incubation: Anti-Flag antibody (purchased from CST, 14793) was diluted at a ratio of 1:1000, and the membrane was incubated with anti-Flag antibody at 4°C overnight, and then the membrane was washed three times with TBST, each time for 5 minutes.

[0278] 6) Secondary antibody incubation: dilute the secondary antibody at a ratio of 1:1000 and incubate the membrane with the secondary antibody at room temperature for 2 hours. Then wash the membrane three times with TBST for 5 minutes each time.

[0279] 7) ECL luminescence detection: Use ECL luminescence solution to detect the luminescence of the membrane.

[0280] Results: As shown in Table 1, using SEQ ID NO: 213 as an example, different nucleoside modifications were performed on the mRNA containing the ORF encoding the uricase fusion protein. Western blotting results showed that HEK293T cells transfected with N1-methylpseudouridine-modified mRNA expressed the highest uricase protein, indicating that N1-methylpseudouridine modification is most suitable for the expression of uricase mRNA in human cells.

[0281] Based on the above experimental results, the subsequent examples all used N1-methylpseudouridine to modify urate oxidase mRNA (SEQ ID NO: 213-380).

[0282] Table 1. Urate oxidase expression levels in lysates of HEK293T cells transfected with mRNA of SEQ ID NO. 213 containing various modified nucleosides

[0283] Example 5. Screening of functional elements of uricase mRNA

[0284] 5.1. mRNA Preparation: The Arthrobacter uricase mRNA (SEQ ID NOs: 213-235) described in Example 1 was prepared using the method described in Example 3, with modification of the mRNA using N1-methylpseudouridine in place of uridine. mRNA was prepared by in vitro transcription and purified and quality-controlled as described in Example 3.

[0285] Results: All N1-methylpseudouridine-modified Arthrococcus urate oxidase mRNAs (SEQ ID NO: 213-235) were detected by agarose gel electrophoresis to have a band of the expected size, and the band was clear without tailing; OD 260nm / OD 280nm All between 2.0 and 2.2, OD260nm / OD 230nm All are between 1.8 and 2.2.

[0286] The above results indicate that all N1-methylpseudouridine-modified Arthritis Bacterium uricase mRNAs (SEQ ID NOs: 213-235) meet the requirements and can be used for subsequent uricase protein expression detection.

[0287] 5.2. Cell transfection: HEK293T cells were transfected with candidate Arthrobacter uricase mRNA (SEQ ID NO: 213-235) modified with N1-methylpseudouridine using the method described in Example 4. After 24 hours of culture, the cell supernatant and cell samples were collected.

[0288] 5.3. Western Blot Detection: The method described in Example 4 was used to detect the expression level of uricase protein in HEK293T cells transfected with candidate Arthrobacter uricase mRNA (SEQ ID NO: 213-235).

[0289] As shown in Table 2, HEK293T cells transfected with different candidate Arthrobacter uricase mRNAs (SEQ ID NOs: 213-235) expressed different levels of uricase protein. Based on the expression levels of uricase, as shown in Table 2, we selected SEQ ID NO. 55 as the preferred 5'-UTR and SEQ ID NO. 61 as the preferred 3'-UTR.

[0290] Table 2. Urate oxidase expression levels in HEK293T cells transfected with candidate mRNAs modified with N1-methylpseudouridine (SEQ ID NOs: 213-235).

[0291] The results of Examples 1-5 indicate that for expressing uricase or its fusion protein in human cells in the form of mRNA sequences, SEQ ID NO: 55 as the 5'-UTR, SEQ ID NO: 61 as the 3'-UTR, and N1-methylpseudouridine as the modified nucleoside are preferred.

[0292] Example 6. Design of mRNA encoding urate oxidase from different species

[0293] To develop a uricase mRNA sequence that is most suitable for expression in human cells and has different secretion efficiencies, the inventors selected uricases from a pool of 21 uricase sequences (SEQ ID NOs: 1-21), including those derived from a human pseudogene (SEQ ID NO: 1), a mammalian (pig-baboon chimeric protein, SEQ ID NO: 2), Aspergillus flavus (SEQ ID NO: 3), Bacillus arthritis (SEQ ID NOs: 4-5), and Candida utilis (SEQ ID NOs: 9-10) as candidate uricase amino acid sequences for developing uricase mRNA therapeutics.

[0294] [Corrected 28.08.2024 in accordance with Rule 26] The inventors introduced signal peptide sequences (SEQ ID NOs:22-43) derived from different proteins into the N-termini of the seven uricase amino acid sequences (SEQ ID NOs:1-5, 9-10) or introduced peroxisomal localization sequences (SEQ ID NOs:46-49) into the N-termini / C-termini of the uricase amino acid sequences to construct various uricase fusion proteins. Subsequently, the coding sequences of wild-type uricase and uricase fusion proteins were codon-optimized to design one or more ORF sequences (SEQ ID NOs:68-201) for each uricase fusion protein. In addition, the inventors combined the preferred 5'-UTR sequence (SEQ ID NO: 55) and preferred 3'-UTR sequence (SEQ ID NO: 61) screened in Examples 1-5 with various other functional elements, following the structure of 5'-cap-5'-UTR coding sequence-codon-optimized nucleotide sequence encoding uricase fusion protein-3'-UTR coding sequence-3'-polyadenylation (as shown in Figure 1). mRNAs (SEQ ID NOs: 236-369) were assembled for each sequence: one encoding a uricase variant without a tag and one encoding a uricase variant with a Flag tag at the C-terminus for subsequent expression verification.

[0295] Example 7. In vitro activity detection of mRNA encoding urate oxidase from different species

[0296] HEPG2 cells were transfected with mRNAs (SEQ ID NOs: 236-369) encoding urate oxidases of different species (SEQ ID NOs: 1-5, 9-10) constructed in the aforementioned examples, and urate oxidase activity in HEPG2 cell lysate samples and cell supernatant samples was detected in vitro.

[0297] 7.1. mRNA Preparation: N1-methylpseudouridine-modified candidate urate oxidase mRNAs (SEQ ID NOs: 236-369) were prepared using the in vitro transcription (IVT) method described above. After in vitro transcription, mRNA was purified and quality tested according to the method described in Example 3.

[0298] Results: Agarose gel electrophoresis of all candidate uricase mRNAs modified with N1-methylpseudouridine (SEQ ID NO: 236-369) showed a band of the expected size, which was clear and had no tail. 260nm / OD 280nm All between 2.0 and 2.2, OD 260nm / OD 230nm All are between 1.8 and 2.2.

[0299] The above experimental results show that all N1-methylpseudouridine-modified uricase mRNAs (SEQ ID NOs: 236-369) meet the requirements and can be used for subsequent uricase activity detection.

[0300] 7.2 Cell Transfection: In this example, HEPG2 cells purchased from ATCC were transfected with N1-methylpseudouridine-modified uricase mRNA (SEQ ID NOs: 236-369) using the transfection method described in Example 4. Urate oxidase activity was then measured in cell lysate samples and / or cell supernatant samples. HEPG2 cells were cultured in MEM medium (Gibco, 41090036) supplemented with 10% fetal bovine serum (Gibco, 10099) and 1% nonessential amino acids (Gibco, 11140050).

[0301] 7.3. 24 hours after cell transfection, cell supernatant samples and cell samples were collected. After collecting the cell supernatant samples, centrifuge at 1000 rpm for 5 minutes, aspirate the supernatant, and discard the pellet. After collecting the cell samples, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and lyse the cells on ice using RIPA containing protease inhibitors. The cells were pelleted for 30 minutes, followed by centrifugation at 12,000 g at 4°C for 15 minutes, and the supernatant was collected.

[0302] 7.4. Use The urate oxidase activity in cell lysate and cell supernatant samples was detected using the Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181). The specific steps are as follows:

[0303] 1) Reagent preparation: Return all required reagents to room temperature before use.

[0304] 2) 10mM Red storage solution: The reagent (0.26 mg) was dissolved in 100 μL DMSO and stored at -20°C in the dark.

[0305] 3) Reaction Buffer stock solution: Add 4 mL of 5X Reaction Buffer stock solution (Component E) to 16 mL of deionized water (dH2O) to prepare 1X Reaction Buffer stock solution.

[0306] 4) Horseradish Peroxidase (HRP) stock solution: Dissolve HRP (Component C) in 200 μL of 1X Reaction Buffer stock solution to prepare a 100 U / mL Horseradish Peroxidase (HRP) stock solution.

[0307] 5) 20 mM H2O2 working solution: 3% H2O2 stock solution (Component D) is prepared by adding dH2O according to the label volume.

[0308] 6) 100 U / mL uricase (Urate Oxidase) Stock Solution: Dissolve uricase (Component F) in 100 μL dH2O to prepare a 100 U / mL uricase stock solution. Freeze at -20°C.

[0309] 7) Urate oxidase (uricase) analysis steps:

[0310] ① Prepare all necessary reagents and standards according to the above instructions;

[0311] ② Dilute the 100 U / mL uricase stock solution 500-fold to prepare a uricase standard curve at 200 mU / mL, 100 mU / mL, 50 mU / mL, 25 mU / mL, 12.5 mU / mL, 6.25 mU / mL, 3.125 mU / mL, 1.562 mU / mL, and 0.78 mU / mL. Use 1X Reaction Buffer stock solution without uricase as a blank control. Use 50 μL for each reaction.

[0312] ③ Add 50 μL of cell lysate sample and / or cell supernatant sample and standard into the corresponding wells.

[0313] ④ Prepare 100 μM solution containing 0.4 U / mL HRP and 1.0 mM uric acid according to the table below. Reagent working solution.

[0314] ⑤ Add 50 μL of 100 μM dapoxetine containing 0.4 U / mL HRP and 1.0 mM uric acid. The reaction was initiated by adding the reagent working solution to each microplate well containing samples and standards. The reactions were incubated at 37°C in the dark.

[0315] ⑥ Measure fluorescence or absorbance in a microplate reader using excitation light at 560 nm and emission light at 590 nm or absorbance at ~560 nm, and read fluorescence and absorbance at 30 minutes.

[0316] ⑦ For each point, background fluorescence or absorbance was corrected by subtracting the value from the no-urate oxidase control.

[0317] 7.5 Experimental Results: As shown in Table 3, HEPG2 cells transfected with different candidate mRNAs modified with N1-methylpseudouridine (SEQ ID NOs: 236-369) produced and / or secreted different levels of active urate oxidase. The results listed in the table are the average of multiple experiments.

[0318] 1) Urate oxidase mRNA (SEQ ID NOs: 245-246, 317, 328, 336) derived from a human pseudogene (SEQ ID NO: 1)

[0319] Among the mRNAs encoding SEQ ID NO: 1, SEQ ID NO: 317, which does not contain a signal peptide, barely induced HEPG2 cells to produce active urate oxidase (urate oxidase activity was less than 1 mU). The remaining sequences containing a signal peptide all induced HEPG2 cells to produce high urate peroxidase activity (greater than 9 mU), and the vast majority of the active urate oxidase produced was secreted extracellularly (>98%). Among them, SEQ ID NO: 336 induced the strongest urate oxidase activity (12.59 mU).

[0320] 2) mRNA encoding pig-baboon fusion uricase (SEQ ID NO: 2) (SEQ ID NO: 241-243, 268, 318, 329, 335, 340, 342, 344, 366)

[0321] Among the mRNAs encoding SEQ ID NO: 2, SEQ ID NOs: 243, 318, and 366, which do not contain a signal peptide, produced low uricase activity (less than 2 mU) and secreted almost no active uricase into the supernatant. The remaining sequences containing a signal peptide were able to secrete most of the active uricase produced extracellularly (>85%). Among them, SEQ ID NO: 340 induced the highest uricase activity (approximately 5.94 mU).

[0322] 3) Urate oxidase mRNA from Aspergillus flavus (SEQ ID NO: 3) (SEQ ID NOs: 238-240, 267, 299-316, 324-325, 330-331, 333, 365, 367)

[0323] Compared with uricase mRNA sequences from other species, the mRNA encoding SEQ ID NO:3 induced higher uricase activity in HEPG2 overall. This may be related to the higher specific activity of the uricase protein itself. Sequences 240, 315, 316, and 365, which lack a signal peptide, induced only <30% of the active uricase to be secreted into the cell, and the total intracellular and extracellular uricase activity was relatively low (<10 mU). All other mRNA sequences containing a signal peptide induced the majority (>90%) of the active uricase to be secreted into the cell. Among all mRNA sequences encoding uricases from Aspergillus flavus, SEQ ID NOs:301-305 and 314 induced the strongest uricase activity, surpassing SEQ ID NO:238 (with a uricase activity of 23.82 mU; the ORF of this sequence (SEQ ID NO:70) is derived from WO2022267086).

[0324] 4) Urate oxidase mRNA from Arthrobacter (SEQ ID NO: 5) (SEQ ID NOs: 236-237, 244, 247-286, 322-323, 326, 334, 357, 359, 368-369)

[0325] Among the mRNAs encoding SEQ ID NO: 5, SEQ ID NOs: 236, 237, 244, 334, and 359, which do not contain a signal peptide, can induce HEPG2 cells to produce high uricase activity (>10 mU) and can secrete approximately 30-50% of the active uricase extracellularly. SEQ ID NO: 359 induced the highest uricase activity (20.22 mU). SEQ ID NOs: 285, 286, and 322, which contain a peroxisomal localization signal, induced high uricase activity while retaining most of the active uricase intracellularly (secretion ratio <30%). All mRNA sequences containing signal peptides (SEQ ID NOs: 247-284, 368-369) were unable to induce HEPG2 cells to produce active uricase.

[0326] Experimental data suggest that peroxisomal localization signals (SEQ ID NOs: 46-48) appear to reduce the secretion ratio of active Bacillus sp. uricasei urate oxidase (from 34.2% to 26.6%, 21.6%, and 25.6%, respectively) without affecting its uricase activity, while peroxisomal localization signal SEQ ID NO: 49 has no significant effect on the secretion ratio.

[0327] 5) Urate oxidase mRNA (SEQ ID NOs: 321, 339, 351, 353, 355, 363) from Arthrobacter (SEQ ID NO: 4)

[0328] Among the mRNA sequences encoding another Arthrobacter uricase (SEQ ID NO: 4), all uricase mRNAs containing a signal peptide (SEQ ID NOs: 339, 351, 353, and 355) failed to successfully induce uricase activity in HEPG2 cells (<0.2 mU). However, SEQ ID NOs: 321 and 363, which did not contain a signal peptide, successfully induced uricase activity in HEPG2 cells of 9.2 mU and 5.7 mU, respectively, with secretion ratios of 32.8% and 39.7%, respectively.

[0329] Experimental data suggest that the signal peptide sequence affects the expression, secretion and / or protein activity of Arthrobacter uricase (SEQ ID NO: 4-5) in HEPG2 cells.

[0330] 6) Urate oxidase mRNA from Candida utilis (SEQ ID NOs: 9-10) (SEQ ID NOs: 287-298, 319-320, 327, 337-338, 345, 347, 349, 358, 360-362)

[0331] Among the mRNAs encoding SEQ ID NO: 9, SEQ ID NOs: 319, 360, and 361, which do not contain a signal peptide, all successfully induced strong uricase activity in HEPG2 cells, and most of the active uricase produced was retained intracellularly (secretion ratio <30%). Among them, SEQ ID NO: 319 produced the strongest uricase activity (approximately 18.6 mU), with a secretion ratio of 17.5%. Other mRNA sequences containing signal peptides (SEQ ID NOs: 287-298, 327, and 337) were unable to induce strong uricase activity in HEPG2 cells (<2 mU).

[0332] Among the mRNAs encoding SEQ ID NO:10, SEQ ID NO:320, which does not contain a signal peptide, produced the strongest uricase activity (approximately 20.8 mU), and the active uricase produced was mainly retained intracellularly, with a secretion ratio of 16.3%. Among the sequences containing a signal peptide, only SEQ ID NO:349 could induce HEPG2 cells to produce a certain amount of uricase activity (approximately 5.2 mU), and the remaining mRNA sequences were unable to induce HEGP2 to produce high uricase activity (<1.5 mU).

[0333] Experimental data suggest that, like the uricase from Arthritis coli (SEQ ID NOs: 4-5), the expression, secretion and / or protein activity of the uricase from Candida utilis (SEQ ID NO: 9) in HEPG2 cells are also affected by the signal peptide sequence.

[0334] In summary, different uricase proteins have distinct properties, which can affect the in vitro and in vivo expression of mRNAs encoding these proteins. For example, mRNAs encoding the human pseudogene-derived uricase (SEQ ID NO:1) and the Aspergillus flavus-derived uricase (SEQ ID NO:3) require a signal peptide sequence to secrete active uricase out of the cell, and the presence of a signal peptide sequence significantly enhances their expression. In contrast, mRNAs encoding the uricases from Arthritis Bacteria (SEQ ID NOs:4-5) and Candida utilis (SEQ ID NOs:9-10) induce high uricase activity in cells without a signal peptide sequence and can secrete a certain proportion of active uricase out of the cell. However, the signal peptide sequence affects the expression, secretion, and / or activity of these two species' uricase proteins. These characteristics provide a foundation for screening candidate uricase mRNA sequences with different secretion ratios, allowing selection of mRNA sequences encoding different uricase proteins based on the desired secretion ratios.

[0335] The inventors classified all mRNAs that can produce high uricase activity in HEPG2 cells into three categories: ① highly secreted uricase mRNA (secretion ratio >90%), such as SEQ ID NOs: 238, 301-305, 314, 328, 336, 344, etc.; ② highly cell-retained uricase mRNA (secretion ratio <30%), such as SEQ ID NOs: 240, 285-286, 315-316, 319-320, 322, etc.; ③ moderately secreted uricase mRNA (secretion ratio >30% but less than 60%), such as SEQ ID NOs: 236-237, 244, 321, 334, 357, 359, etc.

[0336] Table 3. Urate oxidase activity of HEPG2 cells transfected with candidate mRNAs modified with N1-methylpseudouridine (SEQ ID NOs: 236-369)

[0337] Example 8. Preparation of LNP-mRNA Composition

[0338] To achieve effective in vivo delivery of uricase mRNA, lipid-encapsulated uricase mRNA was used to obtain a LNP-mRNA composition. The specific preparation process is as follows:

[0339] 1) Prepare the aqueous phase: Prepare the mRNA working solution with 10 mM sodium citrate solution (purchased from ThermoFisher) at a pH of 3.8 at a concentration of 0.16 mg / mL.

[0340] 2) Prepare the organic phase: 9.6 mg / mL concentration, lipid dissolved in anhydrous ethanol. The molar percentages of the three formulation components are as follows:

[0341] ①LNP preparation 1 (hereinafter referred to as LNP1):

[0342] DLin-MC3-DMA:DSPC:Cholesterol:DMPE-PEG2000=50:10:38.5:1.5;

[0343] ②LNP preparation 2 (hereinafter referred to as LNP2):

[0344] SM102:DSPC:Cholesterol:mPEG-DMG-2k=50:10:38.5:1.5;

[0345] ③LNP preparation 3 (hereinafter referred to as LNP3):

[0346] ALC-0315:DSPC:Cholesterol:ALC-0519=50:10:38.5:1.5;

[0347] 3) preparing the LNP-mRNA composition using a microfluidic chip on a microfluidic liposome encapsulation instrument at a ratio of aqueous phase to organic phase of 3:1 and a total flow rate of 12 mL / min;

[0348] 4) After preparation, add 10 volumes of DPBS (without calcium and magnesium ions) to dilute;

[0349] 5) Use 30KD ultrafiltration tubes (purchased from Millipore) to concentrate to the pre-dilution volume by centrifugation at 3000 g for 30 minutes at 4°C, and then replace the buffer with DPBS to achieve a replacement rate of 99%;

[0350] 6) Prepare a sucrose solution of corresponding concentration using DPBS, and then dilute the LNP-mRNA composition using the sucrose solution with a final concentration of 5%;

[0351] 7) After the LNP-mRNA composition was prepared, the concentration of the LNP-mRNA composition was determined using the Qubit fluorescence method, and the encapsulation efficiency was calculated. The particle size, zeta potential, and polydispersity (PDI) of the LNP-mRNA composition were detected using a particle size analyzer (Malvern, Zetasizer Pro).

[0352] 8) Finally, the LNP-mRNA composition was stored at -80°C.

[0353] Example 9. Uric acid-lowering effect of mRNA composition encoding urate oxidase in hyperuricemic rats

[0354] To further compare the uric acid-lowering effects of uricase mRNA drugs with different secretion ratios in animals, this example selected representative uricase mRNAs of SEQ ID NO: 238, SEQ ID NO: 303 (high secretion), SEQ ID NO: 244 (moderate secretion), and SEQ ID NO: 319 (high cellular retention). Urate oxidase LNP-mRNA compositions were prepared using LNP formulation 1 from Example 8. The uric acid-lowering effects of the different compositions were evaluated in hyperuricemic rats. The evaluation criteria were serum uric acid levels after intravenous injection of the LNP-mRNA composition encoding uricase into the hyperuricemic rats. In this example, the uricase protein drug Rasburicase (SEQ ID NO: 3, purchased from MCE) was used as a positive reference for comparison with the mRNA composition.

[0355] 9.1. LNP-mRNA Drug Preparation:

[0356] The above-mentioned IVT method was used to prepare candidate uricase mRNAs modified with N1-methylpseudouridine (SEQ ID NOs: 238, 244, 303, 319) and uricase mRNA without modified nucleosides (SEQ ID NO: 238), and relevant quality control tests were performed. Subsequently, LNP-mRNA compositions were prepared from the uricase mRNA modified with N1-methylpseudouridine according to the formula of LNP formulation 1 in Example 7. After the LNP-mRNA compositions were prepared, particle size, zeta potential, polydispersity (DP1), and encapsulation efficiency were tested. As shown in Table 4, the particle size, zeta potential, polydispersity, and encapsulation efficiency of the prepared LNP-mRNA compositions all met the requirements, and the LNP-mRNA compositions passed quality control and can be used in subsequent animal experiments.

[0357] Table 4 LNP quality control data of efficacy experiment

[0358] 9.2. In vivo rat experiment: 80 adult male SD rats weighing 170-190g (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., 16 as spares) were housed under SPF conditions and maintained in ventilated cages with a 12-hour light and 12-hour dark cycle. The rats were randomly assigned to two treatment groups (a blank control group of 10 rats and a modeling group of 70 rats) using the BioBook random allocation function to achieve similar group weights and reduce inter-group variation. The day of grouping was designated Day 0. Starting from Day 0, the 70 rats in the modeling group were continuously fed a high-uric acid diet and 0.5% potassium oxonate was added to the drinking water. After 8-10 days of feeding, blood was collected and serum uric acid was tested to determine whether hyperuricemia had formed (a serum uric acid level of not less than 450μmol / mL was considered a successful model). After successful modeling, the rats continued to be fed a high-uric acid diet and 0.25% potassium oxonate was added to the drinking water. Food and water intake were not restricted. In addition, 10 rats (G1) in the blank control group were fed with normal feed and normal drinking water. The rats with successful modeling were randomly divided into 7 groups according to serum uric acid levels and body weight, with 8 rats in each group. The drugs were administered according to the grouped dosing scheme in Table 5. The doses or concentrations mentioned in the experimental scheme are the doses or concentrations of the mRNA in the composition.

[0359] Table 5 Intravenous administration regimen for rat hyperuricemia model

[0360] Before administration, and 1, 4, 8, 24, and 48 hours after administration, all animals were anesthetized with 1-4% isoflurane inhalation for 3-5 minutes. Approximately 300 μL of whole blood was collected from the orbital cavity. After standing at room temperature for 2 hours, the blood was centrifuged at 2000 g for 10 minutes at 4°C to separate the serum. Uric acid levels in the rat serum samples were analyzed using an automated biochemical analyzer.

[0361] 9.3. Analysis of Results: The efficacy results are shown in Figure 3. Before administration, the blood uric acid level in the G1 normal control group was 95.4 μmol / L, and the blood uric acid levels in the G2-G8 groups were all greater than 450 μmol / L, indicating that the hyperuricemia model was successfully established. Rasburicase (SEQ ID NO: 3), a 0.3 mpk protein drug, rapidly reduced the blood uric acid level in hyperuricemic rats to below the normal rat blood uric acid level (from 493 μmol / L to 65 μmol / L) 1 hour after administration. However, due to the short half-life of the protein drug, the blood uric acid level in the G3 group returned to the hyperuricemia level (553 μmol / L) 24 hours after administration. Compared with the G2 model control group, the blood uric acid levels in the G5-G8 groups were lower 24 hours and 48 hours after administration, with the blood uric acid levels in the G5 and G7 groups being relatively lower. The data suggest that a combination of four N1-methylpseudouridine-modified uricase mRNAs (SEQ ID NOs: 238, 244, 303, and 319) with different secretion ratios and liposomes can reduce the blood uric acid level in hyperuricemic rats within 48 hours after administration (by approximately 150-200 μmol / L), at a dose of 1 mpk. The highly secreted mRNA (SEQ ID NOs: 238 and 303) combination showed a stronger uric acid-lowering ability after administration. Furthermore, the blood uric acid levels of rats in the G4 group after administration were almost identical to those in the G2 model control group, suggesting that uricase mRNA without liposome encapsulation and nucleoside modification has little effect on lowering blood uric acid in animals, and that uricase mRNA still needs to be delivered via liposomes to be effective.

[0362] Conclusion: Urate oxidase mRNA requires liposome delivery to exert its urate-lowering effect in animals. Compared to the marketed urate oxidase protein drug rasburicase (SEQ ID NO: 3), the urate oxidase mRNA composition exhibits a more sustained urate-lowering effect.

[0363] Example 10. Construction and screening of highly expressed uricase mRNA sequences

[0364] In order to obtain a uricase mRNA composition with better uric acid-lowering effect, we first further optimized and screened the uricase mRNA sequence.

[0365] [Corrected 28.08.2024 in accordance with Rule 26] Specifically, the inventors selected four albumin signal peptide sequences (including rat albumin signal peptide (SEQ ID NO:36) and humanized rat albumin signal peptide (SEQ ID NO:43-45)), two spacer sequences (SEQ ID NO:50, 53), and uricase amino acid sequences (SEQ ID NO:1, 3), and constructed various uricase fusion proteins based on the albumin signal peptide-spacer-uricase structure. The uricase fusion proteins were then codon-optimized for the coding sequences of wild-type uricase and the uricase fusion proteins, and ORF sequences (SEQ ID NO:202-212) were designed for each uricase fusion protein. In addition, the inventors combined the preferred 5'-UTR sequence (SEQ ID NO: 55) and preferred 3'-UTR sequence (SEQ ID NO: 61) screened in Examples 1-5 with a structure of 5' cap-5'-UTR coding sequence-codon-optimized nucleotide sequence encoding uricase fusion protein-3'-UTR coding sequence-3'-polyadenylation (as shown in Figure 1). These codon-optimized nucleotide sequences were combined with various other functional elements to assemble mRNA (SEQ ID NO: 370-380). For each sequence, two mRNAs were prepared: one encoding a uricase variant without a tag and the other encoding a uricase variant with a Flag tag at the C-terminus for subsequent expression verification.

[0366] The method in the above example was used to prepare candidate uricase mRNAs modified with N1-methylpseudouridine (SEQ ID NOs: 370-380), and then purified and quality tested. HEPG2 cells were then transfected with uricase mRNAs modified with N1-methylpseudouridine (SEQ ID NOs: 238, 314, 336, 370-380) using the transfection method in Example 4, and the cells were transfected with uricase mRNAs modified with N1-methylpseudouridine according to the method in Example 7. The Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181) was used to assay urate oxidase activity in cell lysate samples and / or cell supernatant samples. Urate oxidase activity is shown in Table 6, where the results are averages from multiple experiments.

[0367] 1) Urate oxidase mRNA (SEQ ID NOs: 238, 314, 370-376) derived from Aspergillus flavus (SEQ ID NO: 3)

[0368] [Corrected 28.08.2024 according to Rule 26] Compared with the highly secreted Aspergillus flavus urate oxidase mRNA (SEQ ID NO: 238, 314, urate oxidase activities of 23.82 and 28.12 mU, respectively) preferably without any spacer sequence in Example 7, mRNAs encoding Aspergillus flavus urate oxidase fusion proteins containing rat or humanized rat albumin signal peptide (SEQ ID NO: 36, 43-45)-spacer sequence (SEQ ID NO: 50, 53) (SEQ ID NO: 370-376) can induce HEPG2 cells to produce higher urate oxidase activity (>41 mU), and the secretion ratio is >87%.

[0369] The experimental results suggest that the functional elements encoding the fusion protein of rat or humanized rat albumin signal peptide (SEQ ID NO: 36, 43-45) and spacer sequence (SEQ ID NO: 50, 53) can significantly increase the expression level of mRNA encoding Aspergillus flavus urate oxidase (SEQ ID NO: 3) (about 1.5-2 times) without affecting its secretion ratio.

[0370] 2) Urate oxidase mRNA (SEQ ID NOs: 336, 377-380) derived from a human pseudogene (SEQ ID NO: 1)

[0371] [Corrected 28.08.2024 according to Rule 26] Compared with the highly secreted human pseudogene uricase mRNA (SEQ ID NO: 336, uricase activity of 12.59 mU) without any spacer sequence preferred in Example 7, mRNAs encoding human pseudogene uricase fusion proteins containing rat or humanized rat albumin signal peptide (SEQ ID NOs: 36, 43-45)-spacer sequence (SEQ ID NO: 53) (SEQ ID NOs: 377-380) induced higher uricase activity in HEPG2 production. Among them, mRNA encoding human pseudogene uricase fusion protein containing rat albumin signal peptide (SEQ ID NO: 36)-spacer sequence (SEQ ID NO: 53) (SEQ ID NO: 379) induced the highest uricase activity in HEPG2 production, which was 27.04 mU, with a secretion ratio of 94.88%.

[0372] Experimental results suggest that functional elements encoding a fusion protein of rat or humanized rat albumin signal peptide (SEQ ID NO:36, 43-45) and a spacer sequence (SEQ ID NO:53) can significantly increase the expression level of mRNA encoding the human pseudogene urate oxidase (SEQ ID NO:1) (approximately 1.25-2.2 times). Among them, the functional element encoding a fusion protein of rat albumin signal peptide (SEQ ID NO:36) and a spacer sequence (SEQ ID NO:53) has the highest increase in the expression level of mRNA encoding the human pseudogene urate oxidase (SEQ ID NO:1), approximately 2.2 times.

[0373] Experimental Conclusion: This example screened for an mRNA functional element that can increase protein expression. This high-expression functional element is a nucleic acid sequence encoding a fusion protein of rat or humanized rat albumin signal peptide (SEQ ID NOs: 36, 43-45) and a spacer sequence (SEQ ID NOs: 50, 53). It can significantly increase the expression level of the protein encoded by the highly secreted uricase mRNA without affecting its secretion ratio. Based on this high-expression functional element, this example screened for a group of highly expressed, highly secreted uricase mRNA candidate molecules (SEQ ID NOs: 370-376, 379).

[0374] Table 6 Urate oxidase activity of HEPG2 cells transfected with candidate mRNA modified with N1-methylpseudouridine (SEQ ID NO: 238, 314, 336, 370-380)

[0375] Example 11. Pharmacokinetics and uric acid-lowering effects of mRNA compositions encoding urate oxidase in mice

[0376] To further compare the druggability of uricase mRNA drugs with different secretion ratios, this example selected representative uricase mRNAs of SEQ ID NOs: 238, 303, and 371 (high secretion), SEQ ID NO: 244 (moderate secretion), and SEQ ID NOs: 286 and 319 (high cellular retention), and used the LNP formulation 1 described in Example 8 to form uricase LNP-mRNA compositions.

[0377] The pharmacokinetics of the mRNA composition encoding uricase in mice were tested. The evaluation criteria were to assess the activity of uricase in the serum of mice after intravenous injection of the LNP-mRNA composition encoding uricase, and simultaneously measure the serum uric acid level to evaluate the therapeutic effect of the mRNA composition encoding uricase. In this example, the uricase protein drug Rasburicase (SEQ ID NO: 3, purchased from MCE) and the Arthrobacter uricase protein (SEQ ID NO: 5) were used as positive controls for comparison with the LNP-mRNA composition.

[0378] 11.1. LNP-mRNA Drug Preparation:

[0379] The above-mentioned IVT method was used to prepare candidate uricase mRNAs modified with N1-methylpseudouridine (SEQ ID NOs: 238, 303, 371, 244, 286, 319), and relevant quality control tests were performed. Subsequently, LNP-mRNA compositions were prepared using the formula of LNP preparation 1 in Example 8, using N1-methylpseudouridine modified uricase mRNA. Empty LNPs were also prepared as a reference. After the compositions were prepared, particle size, zeta potential, polydispersity (DP1), and encapsulation efficiency were tested. As shown in Table 7, the particle size, zeta potential, polydispersity, and encapsulation efficiency of the prepared LNP-mRNA compositions all met the requirements, and the LNP-mRNA composition quality control was qualified and could be used for subsequent animal experiments.

[0380] Table 7 LNP quality control data of mouse PK / PD experiment

[0381] 11.2. In vivo experiments in mice: 90 male C57BL / 6 mice weighing approximately 18-22 g and aged 7-8 weeks (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were housed under SPF conditions and kept in ventilated cages under a 12-hour light and 12-hour dark cycle. One day before the start of dosing, all animals were weighed and grouped using StudyDirector™ (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). 108 mice were divided into 10 groups of 9 mice each, and each group was divided into three subgroups of 3 mice each. The “Matched distribution” random grouping method was selected for grouping to ensure that the average weight of each group / subgroup was as close as possible. The day of grouping was defined as day -1. On Day 0, rats were intravenously administered the LNP-mRNA composition, empty LNP, and protein drug. Detailed dosing methods, dosages, and routes of administration are shown in Table 8. The doses or concentrations mentioned in the experimental protocol refer to the doses or concentrations of the mRNA in the composition. Orbital bleeding was performed on mice before dosing and at 1, 3, 6, 12, 24, and 48 hours after dosing. Blood was collected alternately from the three subgroups, and serum was isolated.

[0382] Table 8 Intravenous administration regimen for mouse PK / PD experiment

[0383] 11.3. In vitro testing: Use The urate oxidase activity of mouse serum samples was detected using Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181). The specific operation protocol was as described in Example 7. The uric acid levels of mouse serum samples were analyzed using an automatic biochemical analyzer (purchased from Sysmex, BX3010).

[0384] 11.4. Results Analysis

[0385] 1) Highly secreted mRNA composition

[0386] As shown in Figure 4a, compared with the G1 PBS-administered group and the G2 empty bag-administered group, the serum urate oxidase activity of animals in groups G3-G5 was significantly increased 3 hours after administration, indicating that the highly secreted urate oxidase mRNA (SEQ ID NOs: 238, 303, 371) compositions can successfully induce the expression of serum urate oxidase in mice, among which the SEQ ID NO: 371 composition can induce the highest serum urate oxidase activity (approximately 108.3 mU / ml).

[0387] As shown in Figure 4c, the urate oxidase protein drug rasburicase (SEQ ID NO: 3) can quickly reduce the serum uric acid level of mice to 0 μmol / l after administration, and the serum uric acid of mice returns to normal levels 12 hours after administration. After administration, the highly secreted urate oxidase mRNA (SEQ ID NO: 238, 303, 371) composition can also reduce the serum uric acid level of mice to close to 0 μmol / l, and the maintenance time is longer than that of the protein group. Among them, LNP-SEQ ID NO: 371 has a better uric acid-lowering effect, and the serum uric acid of mice is at a low level (<50 μmol / l) within 48 hours after administration.

[0388] 2) Medium secreted mRNA composition

[0389] As shown in Figure 4b, compared with the G1 PBS-administered group and the G2 empty bag-administered group, the serum urate oxidase activity of the animals in the G6 group was significantly increased 3 and 6 hours after administration, indicating that the medium secretory urate oxidase mRNA (SEQ ID NO: 244) composition can successfully induce the expression of urate oxidase in mouse serum, which was 40.0 mU / ml.

[0390] As shown in Figure 4d, the Arthrobacter uricase protein (SEQ ID NO: 5) rapidly reduced serum uric acid levels in mice to 0 μmol / l after administration, with serum uric acid levels beginning to rise again 24 hours after administration. A composition containing a moderately secreted uricase mRNA (SEQ ID NO: 244) also reduced serum uric acid levels in mice to near 0 μmol / l within 24 hours after administration.

[0391] 3) Highly cell-retaining mRNA composition

[0392] As shown in Figures 4b and 4d, the highly cell-retaining mRNA composition (SEQ ID NOs: 286, 319) did not induce significant serum urate oxidase activity in mice compared to PBS and empty LNPs, but reduced serum uric acid levels in mice within 24 hours after administration. Compared to the highly secreted and moderately secreted mRNA compositions, the highly cell-retaining mRNA composition reduced serum uric acid to a lesser extent.

[0393] 11.5. Summary: Among uricase mRNAs with different secretion ratios and expressions, highly secreted and moderately secreted uricase mRNA compositions demonstrated superior urate-lowering effects in normal mice. Compared to uricase protein drugs (SEQ ID NOs: 3 and 5), highly secreted and moderately secreted uricase mRNA compositions demonstrated longer half-lives and more sustained urate-lowering efficacy. Among the uricase mRNA sequences selected in this example, SEQ ID NOs: 238, 303, 371, and 244 were more preferred.

[0394] Example 12. Effects of different LNP formulations on the uric acid-lowering effect of uricase mRNA compositions in mice

[0395] This example compares the effects of different LNP formulations on the uric acid-lowering effect of a uricase mRNA composition in mice, using SEQ ID NO: 244 as an example. The effect of different LNP formulations on the efficacy of the uricase mRNA composition was evaluated by measuring serum uric acid levels after administration of the uricase-encoding LNP-mRNA composition to mice.

[0396] 12.1. LNP-mRNA Drug Preparation:

[0397] Use above-mentioned IVT method to prepare the SEQ ID NO:244 modified by N1-methyl pseudouridine, and carry out relevant quality control detection.Subsequently, according to the preparation method in Example 8, prepare the LNP-mRNA compositions of three kinds of LNP preparations (LNP preparations 1-3).After composition preparation is completed, carry out particle diameter, Zeta potential, polydispersity (DPI) and encapsulation efficiency detection.As shown in Table 9, the particle diameter, Zeta potential, polydispersity and encapsulation efficiency of the LNP-mRNA compositions prepared all meet the requirements, and the LNP-mRNA compositions quality control is qualified, and can be used for subsequent animal experiments.

[0398] Table 9 LNP quality control data of mouse PK / PD experiment

[0399] 12.2. In vivo mouse experiments: Twelve male C57BL / 6 mice weighing approximately 18-22 g and aged 7-8 weeks (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were housed under SPF conditions and maintained in ventilated cages with a 12-hour light and 12-hour dark cycle. One day before dosing, all animals were weighed and grouped using StudyDirector™ (version 3.1.399.19, StudyLog System, Inc., San Francisco, CA, USA). The 12 mice were divided into four groups of three mice each. The "matched distribution" randomization method was used to ensure that the average weight of each group was as close as possible. On the first day after grouping, the rats were intravenously administered the LNP-mRNA composition. The detailed administration method, dosage, and route of administration are shown in Table 10. The doses or concentrations mentioned in the experimental protocol are the doses or concentrations of the mRNA in the composition. Orbital bleeding was performed on the mice before, 6 hours after, and 24 hours after administration, and serum was isolated.

[0400] Table 10 Dosage regimens for efficacy experiments of different LNP preparations

[0401] 12.3. In vitro testing:

[0402] The uric acid levels of mouse serum samples were analyzed using a fully automatic biochemical analyzer (purchased from Sysmex, BX3010).

[0403] 12.4. Result Analysis:

[0404] As shown in Figure 5, at a dose of 4 mpk, the urate oxidase mRNA encapsulated by the three LNP formulations could reduce the serum uric acid level of mice to 0 at 6 h and 24 h after administration.

[0405] 12.5. Conclusion:

[0406] The experimental results suggest that the three LNP formulations of uricase mRNA compositions have similar uric acid-lowering efficacy in mice. Example 13. Effects of different administration routes on the pharmacokinetics and uric acid-lowering efficacy of uricase mRNA compositions in mice

[0407] This example uses SEQ ID NO: 371 as an example to compare the effects of different administration routes, including intravenous (iv), subcutaneous (sc), and intramuscular (im), on the pharmacokinetics and uric acid-lowering effects of a uricase mRNA composition in mice. The evaluation criteria include measuring serum uricase activity after administration of a LNP-mRNA composition encoding uricase to mice to evaluate the effects of different administration routes on the pharmacokinetics of the uricase mRNA composition, and simultaneously measuring serum uric acid levels to evaluate the effects of different administration routes on the efficacy of the uricase mRNA composition.

[0408] 13.1. LNP-mRNA Drug Preparation:

[0409] Use above-mentioned IVT method to prepare the SEQ ID NO:371 of N1-methyl pseudouridine modification, and carry out relevant quality control detection.Subsequently according to the preparation method in Example 8, use LNP preparation 2 to prepare LNP-mRNA compositions, and synchronously prepare the empty bag LNP of LNP preparation 2 as a control.After compositions preparation is completed, carry out particle diameter, Zeta potential, polydispersity (DPI) and encapsulation efficiency detection.As shown in Table 11, the particle diameter, Zeta potential, polydispersity and encapsulation efficiency of the LNP-mRNA compositions prepared all meet the requirements, and the LNP-mRNA compositions quality control is qualified, and can be used for subsequent animal experiments.

[0410] Table 11 LNP quality control data of comparative experiment of drug delivery

[0411] 13.2. In vivo mouse experiment: 45 male C57BL / 6J mice weighing approximately 18-22 g and aged 7-8 weeks (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were housed under SPF conditions and maintained in ventilated cages with a 12-hour light and 12-hour dark cycle. One day before dosing, all animals were weighed and grouped using StudyDirector™ (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). The 45 mice were divided into 5 groups of 9 mice each, and each group was divided into 3 subgroups of 3 mice each. The "Matched distribution" randomization method was selected for grouping to ensure that the average weight of each group was as close as possible. The mice were dosed on the first day after grouping. The detailed dosing method, dosage, and route of administration are shown in Table 12. The doses or concentrations mentioned in the experimental protocol are the doses or concentrations of mRNA in the composition. The mice were bled by orbital sampling before administration and 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 96 h, and 144 h after administration (blood was collected in turn from three subgroups in each group), and the serum was separated.

[0412] Table 12 Dosage regimen for comparative experiment

[0413] 13.3. In vitro detection: The uric acid levels of mouse serum samples were analyzed using a fully automatic biochemical analyzer (purchased from Sysmex, BX3010).

[0414] 13.4. Experimental results:

[0415] Figure 6 shows the serum uric acid levels of each group of animals after administration. In the intravenous administration group, serum uric acid levels were close to 0 μmol / L within 1-48 hours after administration, returning to baseline 96 hours after administration. In the subcutaneous administration group, serum uric acid levels were close to 0 μmol / L within 12-24 hours after administration, returning to baseline 96 hours after administration. In the intramuscular administration group, serum uric acid levels also decreased significantly within 48 hours after administration. The experimental data suggest that all three administration methods can reduce serum uric acid levels in mice to varying degrees compared to the control group and pre-dose baseline levels.

[0416] 13.5. Conclusion:

[0417] Administration of the urate oxidase mRNA composition to animals through different administration routes can significantly reduce the serum uric acid levels of the animals.

[0418] Example 14. Pharmacodynamic evaluation of different urate oxidase mRNA compositions in hyperuricemia rats

[0419] To further compare the uric acid-lowering effects of different uricase mRNA drugs in animals, this example selected representative highly secreted (SEQ ID NOs: 238, 371) and moderately secreted (SEQ ID NO: 244) uricase mRNAs. LNP preparation 2 in Example 8 was used to form uricase LNP-mRNA compositions, and empty LNPs of LNP preparation 2 were simultaneously prepared as controls. The uric acid-lowering effects of different compositions were evaluated in hyperuricemic rats. The evaluation criteria were to measure serum uric acid levels after intravenous injection of LNP-mRNA compositions encoding uricase in hyperuricemic rats, and to simultaneously measure serum uricase activity in rats after administration to evaluate the pharmacokinetics of the uricase mRNA compositions. In this example, the uricase protein drug Rasburicase (SEQ ID NO: 3, purchased from MCE) was used as a positive reference for comparison with the mRNA compositions.

[0420] 14.1. LNP-mRNA Drug Preparation:

[0421] The above-mentioned IVT method was used to prepare candidate uricase mRNAs modified with N1-methylpseudouridine (SEQ ID NOs: 238, 244, 371), and relevant quality control tests were performed. Subsequently, LNP-mRNA compositions were prepared using the formula of LNP preparation 2 in Example 8, and blank LNPs were simultaneously prepared. After the compositions were prepared, particle size, zeta potential, polydispersity (DP1), and encapsulation efficiency were tested. As shown in Table 13, the particle size, zeta potential, polydispersity, and encapsulation efficiency of the prepared LNP-mRNA compositions all met the requirements, and the LNP-mRNA composition quality control was qualified and could be used for subsequent animal experiments.

[0422] Table 13 LNP quality control data of efficacy experiment in hyperuricemia rats

[0423] 14.2. In vivo experiment in rats: 66 adult male SD rats weighing 170-190g (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., 18 as spares) were raised under SPF conditions and kept in ventilated cages with a 12-hour light and 12-hour dark cycle. The rats were randomly assigned to two treatment groups (6 blank control groups and 60 modeling groups) using the BioBook random allocation function to achieve similar average weights for each group and reduce inter-group deviations. After grouping, the 66 rats in the modeling group were continuously fed with a high uric acid feed and 0.5% potassium oxonate was added to the drinking water. Blood was collected after 8-10 days of feeding, and serum uric acid was tested to determine whether hyperuricemia had formed (a serum uric acid content of not less than 450μmol / mL was considered a successful model). After successful modeling, the rats continued to be fed with a high uric acid feed and 0.25% potassium oxonate was added to the drinking water. Food and water intake were not restricted. In addition, 6 rats (G1) in the blank control group were fed with normal feed and normal drinking water. According to the serum uric acid level and body weight, the rats with successful modeling were randomly divided into 7 groups, with 6 rats in each group. The drugs were administered according to the grouped dosing scheme in Table 14. The doses or concentrations mentioned in the test scheme are the doses or concentrations of mRNA in the composition. Before administration and 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, and 192h after administration, all animals were anesthetized by inhaling 1-4% isoflurane for 3-5min, and about 300μL of whole blood was collected from the orbital cavity. After standing at room temperature for 2 hours, the blood was centrifuged at 4℃2000g for 10min to separate the serum.

[0424] Table 14 Intravenous administration regimen for rat hyperuricemia model

[0425] 14.3. In vitro testing:

[0426] 1) Rat serum samples were analyzed for uric acid levels using a fully automated biochemical analyzer (Sysmex, BX3010). The inventors defined 360 μmol / L as the critical serum uric acid level (as indicated by the dotted line in Figure 7a). A serum uric acid level below 360 μmol / L was considered normal. That is, after administration, a serum uric acid level below 360 μmol / L was considered effective, while a level above 360 ​​μmol / L was considered ineffective or ineffective.

[0427] 2) Use The urate oxidase activity of rat serum samples was detected using Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181). The specific operation protocol is as described in Example 7.

[0428] 14.4. Result Analysis:

[0429] 1) The changes in serum uric acid levels of animals in each group are shown in Figure 7a. Within 192 hours, the serum uric acid of normal rats in group G1 was always less than 200 μmol / l, which is consistent with the normal level of serum uric acid in rats. Before administration, the serum uric acid of animals in groups G2-G8 was greater than 450 μmol / l, indicating that the hyperuricemia rat model was successfully established. The serum uric acid of animals in groups G2-G4 was always greater than 450 μmol / l within 8h-192h after administration. In the group (G3 group) administered with the urate oxidase protein drug rasburicase (SEQ ID NO: 3), no decrease in serum uric acid levels was observed 8 hours after administration. This is most likely because SEQ ID NO: 3 has a short half-life and has lost its effectiveness 8 hours after administration.

[0430] As shown in the G5-G7 groups in Figure 7a, the highly secreted uricase mRNA composition (LNP-SEQ ID NO: 238, 371) can reduce the serum uric acid level of hyperuricemic rats to close to 0 μmol / l. Among them, 3mpk LNP-SEQ ID NO: 238 can reduce the serum uric acid of hyperuricemic rats to close to 0 μmol / l for about 1 day, and to normal levels (<360 μmol / l) for about 3 days. LNP-SEQ ID NO: 371 can reduce the serum uric acid of hyperuricemic rats to close to 0 μmol / l for about 3 days at a dose of 1mpk, and to normal levels for about 5 days; at a dose of 3mpk, it can reduce the serum uric acid of hyperuricemic rats to close to 0 μmol / l for about 6 days, and to normal levels for at least 8 days. The experimental results suggest that the composition of SEQ ID NO: 371 is significantly more effective than SEQ ID NO: 238, and has a lower onset dose.

[0431] As shown in Group G8 in Figure 7a, although the medium secretory uricase mRNA composition (LNP-SEQ ID NO: 244) at a dose of 3 mpk could not reduce the serum uric acid level of hyperuricemic rats to close to 0 μmol / l, it could still reduce the serum uric acid level to below 360 μmol / l after administration, and the lowest serum uric acid mean could be reduced to 203.7 μmol / l, and the serum uric acid level could be reduced to normal levels for about 3 days.

[0432] 2) Changes in serum urate oxidase activity in each group are shown in Figure 7b. The serum urate oxidase activity of rats in groups G1, 2, and 4 remained at the same level as before administration within 192 hours after administration. The serum urate oxidase activity of rats in groups G5-G8 increased to varying degrees after administration.

[0433] As shown in Figure 7b, the highly secreted uricase mRNA compositions (LNP-SEQ ID NO: 238, 371) can significantly increase serum uricase activity. The Cmax of serum uricase activity after administration of 3mpk of LNP-SEQ ID NO: 238 was 263.2mU / mL, and the serum uricase activity returned to baseline values ​​96h after administration. At a dose of 1mpk, the Cmax of serum uricase activity after administration of LNP-SEQ ID NO: 371 was 712.0mU / mL, and the serum uricase activity returned to baseline values ​​96h after administration. At a dose of 3mpk, the Cmax of serum uricase activity after administration was 2058.0mU / mL, and the serum uricase activity returned to baseline values ​​192h after administration. Experimental data suggest that the Cmax of LNP-SEQ ID NO: 371 at doses of 1 and 3 mpk is linear with dose; the efficacy of the highly secreted uricase mRNA composition is consistent with the serum uricase activity after administration.

[0434] As shown in Group G8 in Figure 7b, the serum uricase activity produced after administration of the medium-secreted uricase mRNA composition (LNP-SEQ ID NO: 244) was significantly lower than that of the highly secreted uricase mRNA composition. At a dose of 3 mpk, the serum uricase activity Cmax of the LNP-SEQ ID NO: 244 composition was 21.1 mU / mL, and the serum uricase activity returned to baseline 24 hours after administration. Experimental data suggest that, unlike the highly secreted uricase mRNA composition, after administration of the medium-secreted uricase mRNA composition, even if the serum uricase activity returned to baseline, a certain urate-lowering effect was still maintained, suggesting that the medium-secreted uricase mRNA composition expresses uricase protein in both serum and liver cells to exert its urate-lowering effect.

[0435] 14.5. Summary:

[0436] Combined with the above experimental results, the uricase mRNA composition selected in this example achieved certain pharmacological efficacy in a rat hyperuricemia model induced by a high uric acid diet / potassium oxonate. Among them, the highly secreted uricase mRNA composition LNP-SEQ ID NO: 371 induced high uricase activity in rat serum (Cmax = 712.9 mU / mL) at a relatively low dose (1 mpk); it had a good uric acid-lowering effect, reducing blood uric acid levels to 0 for approximately 3 days and to the normal range for approximately 5 days; it was the most preferred uricase mRNA composition.

[0437] Although the efficacy of the moderately secreted uricase mRNA composition selected in this example is not as good as that of the highly secreted uricase mRNA composition, considering that the hyperuricemia animal model selected in this example is induced by potassium oxonate, which is a uricase inhibitor that rapidly accumulates in the liver after administration to exert its effect, the potassium oxonate-induced hyperuricemia rat model may not be the most suitable model for the moderately secreted uricase mRNA composition that exerts its efficacy by expressing uricase protein in liver cells. Therefore, the inventors will further evaluate the moderately secreted uricase mRNA composition in a hyperuricemia mouse model with uricase gene knockout.

[0438] Example 15. Pharmacodynamic evaluation of different uricase mRNA compositions in the UOX-KO mouse model

[0439] This example uses a mouse model of hyperuricemia with urate oxidase gene knockout (UOX-KO) to further evaluate the uric acid-lowering efficacy of the preferred highly secreted urate oxidase mRNA composition (LNP-SEQ ID NO: 371) and moderately secreted urate oxidase mRNA composition (LNP-SEQ ID NO: 359) in the above examples. A urate oxidase LNP-mRNA composition was prepared using LNP preparation 2 in Example 8 and mRNA, and an empty LNP of LNP preparation 2 was simultaneously prepared as a control. The evaluation criteria were to detect serum uric acid levels after injection of the LNP-mRNA composition encoding urate oxidase in UOX-KO mice, and to simultaneously detect the serum urate oxidase activity of rats after administration to evaluate the pharmacokinetics of the urate oxidase mRNA composition. In this example, the urate oxidase protein drug Rasburicase (SEQ ID NO: 3, purchased from MCE) was used as a positive reference for comparison with the mRNA composition.

[0440] 15.1. LNP-mRNA Drug Preparation:

[0441] The above-mentioned IVT method was used to prepare candidate uricase mRNA (SEQ ID NO: 371, 359) modified with N1-methylpseudouridine, and relevant quality control tests were performed. Subsequently, LNP-mRNA compositions were prepared according to the formula of LNP preparation 2 in Example 8, and blank LNPs were simultaneously prepared. After the composition was prepared, particle size, zeta potential, polydispersity (DP1) and encapsulation efficiency were tested. As shown in Table 15, the particle size, zeta potential, polydispersity and encapsulation efficiency of the prepared LNP-mRNA compositions all met the requirements, and the LNP-mRNA composition quality control was qualified and could be used for subsequent animal experiments.

[0442] Table 15 LNP quality control data of UOX-KO mouse efficacy experiment

[0443] 15.2. In Vivo Mouse Experiments: 65 male UOX-KO mice, 5-6 weeks old, weighing 18-28 grams (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., with 17 mice kept as spares) and 6 male C57BL / 6J mice, 5-6 weeks old, weighing 25-30 grams (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were housed under SPF conditions in ventilated cages with a 12-hour light / 12-hour dark cycle. Blood was collected from the animals 7 days before the start of the experiment (D-7): 80-100 μL of blood was collected from the orbital cavity, and 20-30 μL of serum was separated for serum uric acid analysis. Forty-eight UOX-KO mice with serum uric acid levels between 450 μmol / L and 900 μmol / L were selected for enrollment. Using BioBook, the 48 UOX-KO mice were divided into eight groups of six mice each, using serum uric acid level as the primary indicator and mouse body weight as a secondary indicator. The 9 groups of animals (1 group of C57B / 6J and 8 groups of UOX-KO) with 6 mice in each group were divided into two subgroups, with 3 mice in each subgroup. One week after blood collection (D0), the urate oxidase mRNA composition and protein drug were administered according to the scheme in Table 16. The doses or concentrations mentioned in the experimental scheme are the doses or concentrations of mRNA in the composition. About 100 μL of orbital blood was collected before administration and 8h, 24h, 48h, 96h, 144h, 192h, and 240h after administration (blood was collected from two subgroups of each group of animals in turn). After standing at room temperature for 2 hours, the serum was separated by centrifugation at 4°C 2000g for 10 minutes.

[0444] Table 16 Intravenous administration regimen for efficacy experiment in UOX-KO mice

[0445] 15.3. In vitro testing:

[0446] 1) Uric acid levels in mouse serum samples were analyzed using a blood biochemistry instrument (purchased from Hitachi, 7020). The inventors defined 360 μmol / L as the critical point for serum uric acid levels, with serum uric acid levels below 360 μmol / L considered normal. That is, after administration, a serum uric acid level below 360 μmol / L was considered effective, while a level above 360 ​​μmol / L was considered ineffective or ineffective.

[0447] 2) Use The urate oxidase activity of mouse serum samples was detected using Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181). The specific operation protocol is as described in Example 7.

[0448] 15.4. Experimental results:

[0449] 1) Changes in serum uric acid levels in each group are shown in Figure 8a. Over 240 hours, serum uric acid levels in C57B / 6J mice in group G1 remained below 360 μmol / L, consistent with normal serum uric acid levels in mice. Prior to administration, serum uric acid levels in groups G2-G9 were all above 450 μmol / L, consistent with the levels at enrolment, demonstrating successful establishment of the UOX-KO hyperuricemia mouse model. Serum uric acid levels in groups G2 and G4 remained above 450 μmol / L from 8 to 192 hours after administration. In the group (G3 group) treated with the uricase protein drug rasburicase (SEQ ID NO: 3), the serum uric acid levels of animals were significantly reduced 8 hours after administration (mean: 392.9 μmol / L). Experimental data suggest that the uricase protein drug (SEQ ID NO: 3) can significantly reduce the serum uric acid levels in UOX-KO hyperuricemia mice. The failure to observe a decrease in serum uric acid levels to the normal range is likely due to the short half-life of SEQ ID NO: 3, which began to lose its effectiveness 8 hours after administration.

[0450] Compared with the G2-4 control group, animals (G5-8) administered with different doses of the highly secreted uricase mRNA composition LNP-SEQ ID NO: 371 all showed varying degrees of reduction in serum uric acid. The serum uric acid level in the G8 group decreased less, with serum uric acid levels within the normal range only 48 hours after administration (mean 186.1 μmol / L). The serum uric acid level at the rest of the time was above 360 ​​μmol / L. The serum uric acid level of animals in the G5-7 group decreased to 0 μmol / L after administration. Experimental data suggest that in UOX-KO hyperuricemia mice, the LNP-SEQ ID NO: 371 composition had an unsatisfactory uric acid-lowering effect at a dose of 0.1 mpk. However, at doses of 0.3, 1, and 3 mpk, serum uric acid in UOX-KO hyperuricemia mice was reduced to 0 μmol / L for approximately 2 days, 2 days, and 4 days, respectively, and to the normal range (<360 μmol / L) for approximately 2 days, 4 days, and 4 days, respectively. The experimental results demonstrate that the uric acid-lowering efficacy of the highly secreted uricase mRNA composition (LNP-SEQ ID NO: 371) is dose-dependent, with the minimum effective dose being greater than 0.1 mpk and less than 0.3 mpk.

[0451] Compared to the G2-4 control group, the group of animals (G9) given the moderately secreted uricase mRNA composition LNP-SEQ ID NO: 359 maintained serum uric acid levels within the normal range from 8 hours to 240 hours after administration. Experimental data suggest that although LNP-SEQ ID NO: 359 at a dose of 3 mpk was unable to reduce serum uric acid levels in UOX-KO hyperuricemia mice to 0 μmol / L, it was able to reduce serum uric acid levels to within the normal range (<360 μmol / L) and maintain the therapeutic effect for at least 240 hours. Experimental results show that in the UOX-KO hyperuricemia mouse model, the uric acid-lowering effect of the moderately secreted uricase mRNA composition (LNP-SEQ ID NO: 359) lasted significantly longer than that of the highly secreted uricase mRNA composition (LNP-SEQ ID NO: 371) at the same dose.

[0452] 2) Changes in serum urate oxidase activity in animals of each group are shown in FIG8 b . Animals in groups G1-4 did not show an increase in serum urate oxidase activity from 8 hours to 240 hours after administration.

[0453] A highly secretory uricase mRNA composition (LNP-SEQ ID NO: 371) significantly elevated serum uricase activity in animals at doses of 3, 1, and 0.3 mpk, with Cmax values ​​of 656.03, 254.44, and 104.88 mU / mL, respectively. These values ​​returned to baseline levels 96, 96, and 48 hours after administration, respectively. A dose of 0.1 mpk did not induce significant uricase activity in the animals. Experimental data demonstrated that serum uricase activity after LNP-SEQ ID NO: 371 administration was dose-dependent and positively correlated with urate-lowering efficacy.

[0454] The moderately secreted uricase mRNA composition (LNP-SEQ ID NO: 359) also induced serum uricase activity in UOX-KO mice at a dose of 3 mpk, with a Cmax of 53.05, which occurred 24 hours after administration. 48 hours after administration, the serum uricase activity of animals in the LNP-SEQ ID NO: 359-administered group returned to the baseline level before administration. Experimental data showed that the uric acid-lowering effect of LNP-SEQ ID NO: 359 was not positively correlated with serum uricase activity, suggesting that the moderately secreted uricase mRNA composition expresses uricase protein in both serum and liver cells to exert its uric acid-lowering effect.

[0455] 15.5. Summary:

[0456] 1) The highly secreted uricase mRNA composition LNP-SEQ ID NO: 371 demonstrated consistent, dose-dependent efficacy in both the UOX-KO mouse model and the potassium oxonate-induced hyperuricemia rat model, with the activity positively correlated with serum uricase activity. The minimum effective dose is expected to be between 0.1 and 0.3 mpk.

[0457] 2) The moderately secreted uricase mRNA composition LNP-SEQ ID NO: 359 can reduce the serum uric acid level of UOX-KO hyperuricemia mice to the normal range at a dose of 3 mpk and maintain it for at least 10 days. Its efficacy is not positively correlated with the serum uricase activity induced by the mRNA composition in the animals, suggesting that the moderately secreted uricase mRNA composition LNP-SEQ ID NO: 359 expresses uricase in both serum and liver cells to exert uric acid-lowering efficacy. Therefore, the moderately secreted uricase mRNA composition (such as LNP-SEQ ID NO: 359) can be used as a candidate composition for further development and verification.

[0458] Example 16. Pharmacodynamic evaluation of a moderately secreted uricase mRNA composition at different doses and dosing frequencies in a UOX-KO mouse model

[0459] This example further explores the uric acid-lowering effect of a moderately secreted uricase mRNA composition in UOX-KO mice at different doses and dosing frequencies, using LNP-SEQ ID NO: 359 as an example. A uricase LNP-mRNA composition was prepared using LNP formulation 2 from Example 8 and mRNA. A blank LNP of LNP formulation 2 was simultaneously prepared as a control. The pharmacokinetics of the uricase mRNA composition were evaluated by measuring serum uric acid levels after injection of the uricase-encoding LNP-mRNA composition into UOX-KO mice and simultaneously measuring serum uricase activity in rats after administration.

[0460] 16.1. LNP-mRNA Drug Preparation:

[0461] The above-mentioned IVT method was used to prepare a candidate uricase mRNA (SEQ ID NO: 359) modified with N1-methylpseudouridine, and relevant quality control tests were performed. Subsequently, an LNP-mRNA composition was prepared using the formula of LNP preparation 2 in Example 8, and blank LNPs were simultaneously prepared. After the composition was prepared, particle size, zeta potential, polydispersity (DP1), and encapsulation efficiency were tested. As shown in Table 17, the particle size, zeta potential, polydispersity, and encapsulation efficiency of the prepared LNP-mRNA composition all met the requirements, and the LNP-mRNA composition passed quality control and can be used for subsequent animal experiments.

[0462] Table 17 LNP quality control data of UOX-KO mouse efficacy experiment

[0463] 16.2. In Vivo Mouse Experiments: Fifty male UOX-KO mice, 5-6 weeks old, weighing 18-28 grams (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., with eight mice kept as spares) and six male C57BL / 6J mice, 5-6 weeks old, weighing 25-30 grams (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were housed in SPF conditions and maintained in ventilated cages with a 12-hour light / 12-hour dark cycle. Seven days before the start of the experiment (D-7), blood was collected from the animals: 80-100 μL of blood was collected from the orbital cavity, and 20-30 μL of serum was separated for serum uric acid analysis. Forty-two UOX-KO mice with serum uric acid levels between 450 μmol / L and 900 μmol / L were selected for enrollment. Using BioBook, the 42 UOX-KO mice were divided into seven groups of six mice each, using serum uric acid level as the primary indicator and mouse body weight as a secondary indicator. The 6 mice in each of the 8 groups of animals (1 group of C57B / 6J and 7 groups of UOX-KO) were divided into two subgroups, with 3 mice in each subgroup. One week after blood collection (D0), the urate oxidase mRNA composition was administered according to the scheme in Table 18. The doses or concentrations mentioned in the experimental scheme are the doses or concentrations of mRNA in the composition. About 100 μL of orbital blood was collected before administration and 8h, 24h, 72h, 120h, 168h, 240h, 312h, and 384h after administration (blood was collected from two subgroups of each group of animals in turn). After standing at room temperature for 2 hours, the serum was separated by centrifugation at 4°C 2000g for 10 minutes.

[0464] Table 18 Intravenous administration regimen for efficacy experiment in UOX-KO mice

[0465] 16.3. In vitro testing:

[0466] 1) Uric acid levels in mouse serum samples were analyzed using a blood biochemistry instrument (purchased from Hitachi, 7020). The inventors defined 360 μmol / L as the critical point for serum uric acid levels, with serum uric acid levels below 360 μmol / L considered normal. That is, after administration, a serum uric acid level below 360 μmol / L was considered effective, while a level above 360 ​​μmol / L was considered ineffective or ineffective.

[0467] 2) Use The urate oxidase activity of mouse serum samples was detected using Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181). The specific operation protocol is as described in Example 7.

[0468] 16.4. Experimental results:

[0469] 1) Changes in serum uric acid levels in each group of animals are shown in Figure 9a. Within 384 hours after administration, the serum uric acid level of C57B / 6J mice in the G1 group was always below 360 μmol / L, which is consistent with the normal level of serum uric acid in mice. Before administration, the serum uric acid levels of animals in the G2-G8 groups were all greater than 450 μmol / L, which was consistent with the blood uric acid levels at the time of blood sampling. The UOX-KO hyperuricemia mouse model was successfully established. The serum uric acid levels of animals in the G2-3 groups were always greater than 450 μmol / L within 8h-384h after administration. Compared with the G2-3 control group, animals (G4-8) given the medium secretory urate oxidase mRNA composition LNP-SEQ ID NO: 359 all showed varying degrees of serum uric acid reduction. Among them, although the serum uric acid of animals in the G4 group was significantly reduced after administration, it was always above 360 ​​μmol / L; the serum uric acid of animals in the G7 group dropped to 0 μmol / L within 8-24 hours after administration. Experimental data suggest that in UOX-KO hyperuricemia mice, the LNP-SEQ ID NO: 359 composition has an unsatisfactory uric acid-lowering effect at a single dose of 0.5 mpk; at single doses of 1 and 3 mpk, the serum uric acid level of UOX-KO mice can be reduced to the normal range (<360 μmol / L) and maintained for about 5 days and 10 days, respectively; at a single dose of 6 mpk, the serum uric acid level of UOX-KO mice can be reduced to 0 μmol / L for about 1 day, and then to the normal range (<360 μmol / L) and maintained for about 13 days; a dosing regimen of 1 mpk q2d*3 can reduce the serum uric acid level of UOX-KO mice to the normal range and maintain it for about 13 days.

[0470] Experimental results demonstrated that the uric acid-lowering efficacy of a moderately secreted uricase mRNA composition (LNP-SEQ ID NO: 359) was dose-dependent, with the lowest effective dose being greater than 0.5 mpk and less than or equal to 1 mpk. Compared to a single dose of 3 mpk, a 1 mpk regimen every two days for three days demonstrated greater uric acid-lowering efficacy.

[0471] 2) Changes in serum urate oxidase activity in each group of animals are shown in Figure 9b. Animals in groups G1-3 and 7 did not show significant increases in serum urate oxidase activity after administration. A single dose of 1, 3, and 6 mpk of the moderately secreted urate oxidase mRNA composition (LNP-SEQ ID NO: 359) transiently elevated serum urate oxidase activity in animals, with Cmax values ​​of 21.96, 35.65, and 33.18 mU / mL, respectively. These values ​​returned to baseline levels 72, 72, and 120 hours after administration, respectively. A dose of 0.5 mpk did not induce significant serum urate oxidase activity in animals.

[0472] Experimental data showed that the linear relationship between the serum urate oxidase activity of animals after administration of LNP-SEQ ID NO: 359 and the dosage was not significant, and was not positively correlated with the uric acid-lowering efficacy.

[0473] 16.5. Summary:

[0474] Compared to the highly secreted uricase mRNA composition, the moderately secreted uricase mRNA composition LNP-SEQ ID NO:359 demonstrated a longer-lasting uric acid-lowering effect in the UOX-KO mouse model, but at a higher minimum effective dose, estimated to be between 0.5 and 1 mpk. The efficacy of LNP-SEQ ID NO:359 was positively correlated with the dose administered, but not with the serum uricase activity induced by the mRNA composition in the animals, suggesting that the moderately secreted uricase mRNA composition LNP-SEQ ID NO:359 expressed uricase in both serum and liver cells to exert its uric acid-lowering effect. Compared to a single dose of 3 mpk, a 1 mpk q2d*3 administration regimen was more effective in lowering uric acid.

[0475] Example 17. Pharmacokinetic and pharmacodynamic evaluation of highly secreted uricase mRNA composition in normal rats

[0476] This example further compares the pharmacokinetics and pharmacodynamics of a highly secreted uricase mRNA composition using LNP-SEQ ID NO: 371 with the corresponding uricase protein drug, rasburicase (SEQ ID NO: 3), in normal rats. The evaluation criteria were serum uricase activity and serum uric acid levels measured after intravenous administration to normal rats.

[0477] 17.1. LNP-mRNA Drug Preparation:

[0478] The above-mentioned IVT method was used to prepare a candidate uricase mRNA (SEQ ID NO: 371) modified with N1-methylpseudouridine, and relevant quality control tests were performed. Subsequently, an LNP-mRNA composition was prepared using the formula of LNP preparation 2 in Example 8 to modify the uricase mRNA modified with N1-methylpseudouridine, and blank LNPs were simultaneously prepared. After the composition was prepared, particle size, zeta potential, polydispersity (DP1), and encapsulation efficiency were tested. As shown in Table 19, the particle size, zeta potential, polydispersity, and encapsulation efficiency of the prepared LNP-mRNA composition all met the requirements, and the LNP-mRNA composition quality control was qualified and could be used for subsequent animal experiments.

[0479] Table 19 LNP quality control data of normal rat PK / PD experiment

[0480] 17.2. In vivo experiment in rats: 27 6-week-old male SD rats weighing approximately 230-280 g (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were raised under SPF conditions and kept in ventilated cages under a 12-hour light and 12-hour dark cycle. One day before the start of dosing, all animals were weighed and grouped using StudyDirector™ (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). The 27 rats were divided into 9 groups of 3 rats each. The “Matched distribution” random grouping method was selected for grouping to ensure that the average weight of each group was as close as possible. The rats were dosed on the first day after grouping. The detailed dosing method, dosage and route of administration are shown in Table 20. The doses or concentrations mentioned in the experimental protocol are the doses or concentrations of mRNA in the composition. About 250 μL of whole blood was collected from the rats through the jugular vein before administration and 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 96 h, and 144 h after administration, and 70-80 μL of serum was separated.

[0481] Table 20 Dosage regimen for PK / PD experiment in normal rats

[0482] 17.3. In vitro testing:

[0483] use The urate oxidase activity of rat serum samples was detected using Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181), and the specific operation protocol was as described in Example 7. The uric acid levels of rat serum samples were analyzed using a fully automatic biochemical analyzer (purchased from Hitachi, 7020).

[0484] 17.4. Experimental results:

[0485] 1) Serum urate oxidase activity in each group of animals after administration is shown in Figures 10a-b. Group G3-4, which received the protein drug rasburicase (SEQ ID NO: 3), showed elevated serum urate oxidase activity after administration, with average Cmax values ​​of 169.25 and 58.83 mU / mL at doses of 0.3 and 0.5 mpk, respectively. Serum urate oxidase activity returned to baseline levels 6 and 12 hours after administration, respectively.

[0486] G5-9 is a group that received different doses of LNP-SEQ ID NO: 371, in which LNP-SEQ ID NO: 371 was diluted to different concentrations using PBS. At doses of 1, 0.8, 0.6, and 0.4 mpk, the animals' serum urate oxidase activity reached Cmax 12 hours after administration, reaching 389.5, 318.37, 208.78, and 73.83 mU / mL, respectively. However, after administration of 0.2 mpk of LNP-SEQ ID NO: 371, there was no significant increase in serum urate oxidase activity. Experimental data suggest that the Cmax of serum urate oxidase activity in rats after LNP-SEQ ID NO: 371 administration is dose-dependent.

[0487] As shown in Figure 10c, when the serum urate oxidase activity Cmax after administration was consistent, the half-life of the highly secreted uricase mRNA composition LNP-SEQ ID NO: 371 and the corresponding uricase protein drug SEQ ID NO: 3 (Group G4 and Group G8) was compared. The half-life of the mRNA composition was significantly longer than that of the protein drug.

[0488] 2) Serum uric acid levels in each group of animals after administration are shown in Figures 11a-b. At doses of 0.5 and 0.3 mpk, serum uric acid levels rapidly decreased to approximately 20 μmol / L one hour after administration of the protein drug rasburicase (SEQ ID NO: 3), and then rebounded 12 hours after administration.

[0489] The LNP-SEQ ID NO: 371 composition can reduce the serum uric acid level of rats to approximately 20 μmol / L at different doses of 0.2-1 mpk. The duration of its efficacy is positively correlated with the dosage and the serum urate oxidase activity.

[0490] As shown in Figure 11c, when the serum urate oxidase activity Cmax after administration was consistent, the duration of efficacy of the highly secreted uricase mRNA composition LNP-SEQ ID NO: 371 and the corresponding uricase protein drug SEQ ID NO: 3 (Groups G4 and G8) was compared. The duration of efficacy of the mRNA composition was significantly longer than that of the protein drug.

[0491] 17.5. Summary:

[0492] This example uses LNP-SEQ ID NO: 371 as an example to demonstrate that the uric acid-lowering effect of a highly secreted uricase mRNA composition, serum uricase activity, and dosage in normal rats are positively correlated. Furthermore, it was demonstrated that, when the Cmax of serum uricase activity after administration remains consistent, the highly secreted uricase mRNA composition exhibits a longer half-life and better efficacy than the corresponding uricase protein.

[0493] Example 18. Pharmacokinetic and pharmacodynamic evaluation of different highly secreted uricase mRNA compositions in normal mice

[0494] In this example, different highly secreted uricase mRNAs (SEQ ID NOs: 371, 373, 375-376) were combined with the liposome preparation 2 described in Example 8 to form uricase mRNA compositions. The pharmacokinetics and pharmacodynamics of these compositions in mice were evaluated. The evaluation criteria were serum uricase activity and serum uric acid levels after intravenous administration to normal mice.

[0495] 18.1. LNP-mRNA Drug Preparation:

[0496] The above-mentioned IVT method was used to prepare candidate uricase mRNAs modified with N1-methylpseudouridine (SEQ ID NOs: 371, 373, 375-376), and relevant quality control tests were performed. Subsequently, LNP-mRNA compositions were prepared using the formula of LNP preparation 2 in Example 8, and blank LNPs were simultaneously prepared. After the compositions were prepared, particle size, zeta potential, polydispersity (DP1), and encapsulation efficiency were tested. As shown in Table 21, the particle size, zeta potential, polydispersity, and encapsulation efficiency of the prepared LNP-mRNA compositions all met the requirements, and the LNP-mRNA composition quality control was qualified and could be used for subsequent animal experiments.

[0497] Table 21 LNP quality control data of normal rat PK / PD experiment

[0498] 18.2. In vivo mouse experiment: 54 male C57BL / 6J mice weighing approximately 18-22 g and aged 7-8 weeks (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were housed under SPF conditions and maintained in ventilated cages with a 12-hour light and 12-hour dark cycle. One day before dosing, all animals were weighed and grouped using StudyDirector™ (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). The 54 mice were divided into 6 groups of 9 mice each, each group was divided into 3 subgroups of 3 mice each. The "Matched distribution" randomization method was selected for grouping to ensure that the average weight of each group was as close as possible. The mice were dosed on the first day after grouping. The detailed dosing method, dosage, and route of administration are shown in Table 22. The doses or concentrations mentioned in the experimental protocol are the doses or concentrations of the mRNA in the composition. The mice were bled by orbital sampling before administration and 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 96 h, and 144 h after administration (blood was collected in turn from three subgroups in each group), and the serum was separated.

[0499] Table 22 Dosage regimen for comparative experiment

[0500] 18.3. In vitro testing:

[0501] use The urate oxidase activity of rat serum samples was detected using Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181), and the specific operation protocol was as described in Example 7. The uric acid level of mouse serum samples was analyzed using an automatic biochemical analyzer (purchased from Sysmex, BX3010).

[0502] 18.4. Experimental results:

[0503] 1) Serum urate oxidase activity in each group of animals after administration is shown in Figure 12a. Within 144 hours after administration, the serum urate oxidase activity in the control groups G1-2 remained at baseline levels, while the serum urate oxidase activity in the animals in the G3-6 groups increased significantly, reaching Cmax of 372.55, 733.53, 522.63, and 466.85, respectively, 24 hours after administration. These activities returned to baseline levels 96 hours after administration. These experimental data suggest that the highly secreted serum urate oxidase mRNA compositions LNP-SEQ ID NO: 373, 375, and 376 induced higher serum urate oxidase activity in mice than LNP-SEQ ID NO: 371.

[0504] 2) The serum uric acid levels of the animals in each group after administration are shown in Figure 12b. Within 3-48 hours after administration, the four highly secreted uricase mRNA compositions were able to reduce the serum uric acid levels of the mice to close to 0 μmol / L. The serum uric acid levels returned to the baseline level 96 hours after administration.

[0505] 18.5. Conclusion:

[0506] The experimental results suggest that at the same dosage, LNP-SEQ ID NO: 373, 375, and 376 can induce higher serum urate oxidase protein activity in mice than LNP-SEQ ID NO: 371. Therefore, the minimum effective dose of LNP-SEQ ID NO: 373, 375, and 376 will be lower than that of LNP-SEQ ID NO: 371.

[0507] Example 19. Construction and screening of mRNA sequences encoding half-life-extended uricase

[0508] To obtain a half-life-extended uricase, the inventors selected two half-life-extending elements (including: human VLK sequence SEQ ID NO: 381, human IgG4 Fc sequence SEQ ID NO: 382) and uricase protein from Aspergillus flavus (SEQ ID NO: 3) through a linker (SEQ ID NO: 383) to form a fusion protein. The half-life-extending element can be located at the N-terminus or C-terminus of the uricase. Subsequently, a humanized rat albumin signal peptide (SEQ ID NO: 45) and a spacer sequence (SEQ ID NO: 53) were introduced at the N-terminus of the fusion protein to form a half-life-extended uricase fusion protein.

[0509] [Corrected 28.08.2024 in accordance with Rule 26] The coding sequences of the half-life extended uricase fusion proteins were codon-optimized, and an ORF sequence (SEQ ID NOs:386-389) was designed for each half-life extended uricase fusion protein. Subsequently, the inventors combined the 5'-UTR sequence (SEQ ID NO:384), the 3'-UTR sequence (SEQ ID NO:385), and the poly A tail sequence (SEQ ID NO:64) with various other functional elements to form mRNA (SEQ ID NOs:390-393), following the structure of 5'-UTR coding sequence-codon-optimized nucleotide sequence encoding the half-life extended uricase fusion protein-3'-UTR coding sequence-3'-polyadenylation (as shown in FIG1 ).

[0510] The method in the above example was used to prepare N1-methylpseudouridine modified half-life extended uricase mRNA (SEQ ID NO: 390-393), and was purified and quality tested. HEPG2 cells were then transfected with N1-methylpseudouridine modified half-life extended uricase mRNA (SEQ ID NO: 390-393) using the transfection method in Example 4. Cell lysate samples and cell supernatant samples were collected 72 hours after transfection and used according to the method in Example 7. The Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181) was used to assay urate oxidase activity in cell lysate and cell supernatant samples. Urate oxidase activity is shown in Table 23, where the results are averages from multiple experiments.

[0511] Experimental data showed that 72 hours after transfection, mRNA encoding a half-life-extended uricase fusion protein induced uricase activity in HEPG2 cells, with high secretion rates exceeding 90%. Sequences 391 and 392 induced higher uricase activity in HEPG2 cells, but both were weaker than the highly secreted uricase mRNA sequence, SEQ ID NO: 371, which lacks the half-life-extending element.

[0512] Table 23 Urate oxidase activity of HEPG2 cells transfected with N1-methylpseudouridine modified half-life extended uricase mRNA (SEQ ID NO: 390-393)

[0513] Example 20. Pharmacokinetic and pharmacodynamic evaluation of mRNA compositions encoding half-life-extended uricase fusion proteins in normal rats

[0514] In this example, two half-life-extended uricase mRNAs (SEQ ID NOs: 391-392) and a highly secreted uricase mRNA without a half-life-extending element (SEQ ID NO: 371) selected from Example 19 were combined with the LNP formulation 2 from Example 8 to form mRNA compositions. The pharmacokinetics and pharmacodynamics of the mRNA compositions encoding the half-life-extended uricase fusion proteins were further evaluated in normal rats.

[0515] 20.1. LNP-mRNA Drug Preparation:

[0516] The above-mentioned IVT method was used to prepare candidate uricase mRNA (SEQ ID NO: 371, 391-392) modified with N1-methylpseudouridine, and relevant quality control tests were performed. Subsequently, according to the formula of LNP preparation 2 in Example 8, the uricase mRNA modified with N1-methylpseudouridine was prepared into LNP-mRNA compositions, and blank LNPs were simultaneously prepared. After the compositions were prepared, particle size, zeta potential, polydispersity (DP1) and encapsulation efficiency were tested. As shown in Table 24, the particle size, zeta potential, polydispersity and encapsulation efficiency of the prepared LNP-mRNA compositions all met the requirements, and the LNP-mRNA compositions were qualified in quality control and could be used for subsequent animal experiments.

[0517] Table 24 Half-life extension of urate oxidase mRNA in normal rats PK / PD experiment LNP quality control data

[0518] 20.2. In vivo experiment in rats: 18 6-week-old male SD rats weighing approximately 230-280 g (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were raised under SPF conditions and kept in ventilated cages under a 12-hour light and 12-hour dark cycle. One day before the start of dosing, all animals were weighed and grouped using StudyDirector™ (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). 18 rats were divided into 6 groups of 3 rats in each group. The “Matched distribution” random grouping method was selected for grouping to ensure that the average weight of each group was as close as possible. The rats were dosed on the first day after grouping. The detailed method of administration, dosage and route of administration are shown in Table 25. The doses or concentrations mentioned in the experimental protocol are the doses or concentrations of mRNA in the composition. Before administration, and at 3, 8, 24, 48, 96, 144, 192, and 240 hours after administration, approximately 250 μL of whole blood was collected from the rats through the jugular vein, and 70-80 μL of serum was separated. The animals were weighed regularly during the experiment.

[0519] Table 25 Dosage regimen for half-life extension of urate oxidase mRNA in normal rats PK / PD experiment

[0520] 20.3. In vitro testing:

[0521] 1) Use The urate oxidase activity of rat serum samples was detected using Red Uric Acid / Uricase Assay Kit (purchased from Thermo, A22181). The specific operation protocol is as described in Example 7.

[0522] 2) The uric acid levels of rat serum samples were analyzed using a fully automatic biochemical analyzer (purchased from Hitachi, 7020).

[0523] 20.4. Experimental results:

[0524] 1) Pharmacokinetic evaluation:

[0525] The serum uricase activity of each group of animals after administration is shown in Figure 13a, and the pharmacokinetic parameters of uricase activity are shown in Table 26. Experimental data suggest that at the same dose (1 mpk), the Cmax, AUC, and half-life of the mRNA compositions encoding the half-life-extended uricase fusion proteins, LNP-SEQ ID NO: 391 and 392, were significantly increased compared to the highly secreted uricase mRNA composition, LNP-SEQ ID NO: 371, which does not contain a half-life-extending element. In addition, at a dose of 0.3 mpk, LNP-SEQ ID NO: 391 exhibited a higher AUC and half-life than LNP-SEQ ID NO: 371 at 1 mpk.

[0526] Table 26 Pharmacokinetic parameters of serum urate oxidase activity in rats after administration of mRNA compositions encoding half-life extended urate oxidase fusion proteins

[0527] 2) Pharmacodynamic evaluation:

[0528] Serum uric acid levels in each group of animals after administration are shown in Figure 13b. After administration of the uricase mRNA composition, the serum uric acid levels of rats decreased significantly. At a dose of 1 mpk, the highly secreted uricase mRNA composition LNP-SEQ ID NO: 371, which does not contain a half-life extension element, significantly reduced serum uric acid levels in rats within 3-48 hours after administration. Serum uric acid began to rise 96 hours after administration and returned to baseline 144 hours after administration. The mRNA compositions encoding half-life extended uricase fusion proteins LNP-SEQ ID NO: 391 and 392 significantly reduced serum uric acid levels in rats within 3-96 hours after administration. Serum uric acid levels began to rise 144 hours after administration and returned to baseline 192 hours after administration. In addition, the uric acid-lowering effect and duration of LNP-SEQ ID NO: 391 at a dose of 0.3 mpk were consistent with those of LNP-SEQ ID NO: 371 at 1 mpk. Experimental data showed that at the same dose, the mRNA compositions encoding half-life-extended uricase fusion proteins, LNP-SEQ ID NO:391 and 392, showed a longer duration of efficacy than the mRNA composition LNP-SEQ ID NO:371, which does not contain a half-life-extending element. Furthermore, LNP-SEQ ID NO:391 and 392 exhibited a lower minimum effective dose.

[0529] 20.5. Summary:

[0530] This example validated the two half-life-extended uricase mRNA compositions, LNP-SEQ ID NOs: 391 and 392, through in vivo pharmacokinetic and pharmacodynamic evaluations in normal rats. Compared to the highly secreted uricase mRNA composition, LNP-SEQ ID NO: 371, which does not contain a half-life-extending element, these compositions demonstrated higher serum uricase activity Cmax and AUC, longer half-life and duration of urate-lowering effects, and a lower minimum effective dose after administration.

Claims

1. A nucleic acid comprising an open reading frame (ORF) encoding uricase, a functional fragment or variant thereof.

2. The nucleic acid of claim 1, wherein the functional fragment or variant of uricase is a truncated uricase or a uricase fusion protein that retains uricase activity.

3. The nucleic acid of claim 1 or 2, wherein the uricase comprises the amino acid sequence of any one of SEQ ID NOs: 1-21, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-21.

4. The nucleic acid of any one of claims 1 to 3, wherein the uricase is a uricase derived from a human pseudogene, a mammal (such as a pig, a baboon, a rat, a mouse, a rhesus monkey), an Aspergillus flavus, an arthrocleobacterium, or a Candida utilis, or a chimeric protein of the above uricase.

5. The nucleic acid of any one of claims 1-4, wherein the uricase comprises the amino acid sequence of any one of SEQ ID NOs: 1-5 and 9-10, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-5 and 9-10.

6. The nucleic acid according to any one of claims 1 to 5, wherein the uricase fusion protein is a fusion protein of a signal peptide and an optional spacer sequence with uricase, or a fusion protein of a peroxisome localization peptide with uricase.

7. The nucleic acid according to any one of claims 1 to 6, wherein the uricase fusion protein is a fusion protein comprising a signal peptide, a spacer sequence and uricase from the N-terminus to the C-terminus.

8. The nucleic acid of any one of claims 1 to 7, wherein the uricase fusion protein is a fusion protein comprising a signal peptide, a spacer sequence, a half-life extending element, an optional linker, and uricase from the N-terminus to the C-terminus, a fusion protein comprising a signal peptide, a spacer sequence, a uricase oxidase, an optional linker, and a half-life extending element from the N-terminus to the C-terminus, a fusion protein comprising a signal peptide, a half-life extending element, an optional linker, and uricase from the N-terminus to the C-terminus, or a fusion protein comprising a signal peptide, a uricase oxidase, an optional linker, and a half-life extending element from the N-terminus to the C-terminus. The fusion protein of the present invention comprises a signal peptide, a spacer sequence, a half-life extension element, a linker and a fusion protein of uricase, and optionally, the uricase fusion protein comprises a signal peptide, a spacer sequence, a half-life extension element, a linker and a fusion protein of uricase from the N-terminus to the C-terminus, a signal peptide, a spacer sequence, uricase, a linker and a half-life extension element from the N-terminus to the C-terminus, a signal peptide, a half-life extension element, a linker and a fusion protein of uricase from the N-terminus to the C-terminus, or a signal peptide, uricase, a linker and a half-life extension element from the N-terminus to the C-terminus.

9. The nucleic acid of any one of claims 6-8, wherein the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 22-45, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 22-45.

10. The nucleic acid of any one of claims 6-9, wherein the spacer sequence comprises the amino acid sequence of any one of SEQ ID NOs: 50-53 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 50-53.

11. The nucleic acid of any one of claims 6-10, wherein the half-life extension element comprises SEQ ID NO: 381 or 382, ​​or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 381 or 382.

12. The nucleic acid of claims 6-11, wherein the linker comprises SEQ ID NO: 383 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:

383.

13. The nucleic acid of claim 6, wherein the peroxisome localization peptide comprises the amino acid sequence of any one of SEQ ID NOs: 46-49, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 46-49.

14. The nucleic acid of claim 6 or 13, wherein the peroxisome localization peptide is fused to the N-terminus or C-terminus of the uricase.

15. The nucleic acid of any one of the preceding claims, wherein the ORF is codon-optimized.

16. A nucleic acid according to any of the preceding claims, wherein the ORF is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 67-212, 386-389, or their corresponding DNA sequences.

17. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid further comprises a 5'-untranslated region (5'-UTR).

18. The nucleic acid of claim 17, wherein the 5'-UTR comprises any one of SEQ ID NOs: 54-57, 384, or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 54-57, 384, or a DNA sequence corresponding thereto.

19. The nucleic acid of claim 17 or 18, wherein the 5'-UTR comprises SEQ ID NO: 55, 56 or 384, or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 55, 56 or 384, or a DNA sequence corresponding thereto.

20. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid further comprises a 3'-untranslated region (3'-UTR).

21. The nucleic acid of claim 20, wherein the 3'-UTR comprises any one of SEQ ID NOs: 58-62, 385, or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 58-62, 385, or a DNA sequence corresponding thereto.

22. The nucleic acid of claim 20 or 21, wherein the 3'-UTR comprises SEQ ID NO: 59, 61 or 385, or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 59, 61 or 385, or a DNA sequence corresponding thereto.

23. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid comprises, from 5' to 3', a 5'-UTR, an ORF encoding uricase, a functional fragment or variant thereof, a 3'-UTR, and a 3'-polyadenylation sequence, wherein the ORF is selected from any one of SEQ ID NOs: 67-212, 386-389.

24. The nucleic acid of claim 23, wherein the 5'-UTR is selected from any one of SEQ ID NOs: 54-57, 384, and the 3'-UTR is selected from any one of SEQ ID NOs: 58-62, 385.

25. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid further comprises a 3'-poly A sequence.

26. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid is DNA or mRNA.

27. A nucleic acid according to any one of the preceding claims, comprising at least one chemical modification.

28. The nucleic acid of claim 27, wherein the chemical modification is selected from one or more of 5-methylcytidine, 5-methoxyuridine, pseudouridine, N6-methyladenosine, or N1-methylpseudouridine.

29. The nucleic acid of claim 27 or 28, wherein the chemical modification is N1-methylpseudouridine.

30. A nucleic acid according to any of the preceding claims, wherein at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or 100% of the uridine in the nucleic acid is chemically modified to N1-methylpseudouridine.

31. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid is an mRNA and the mRNA further comprises a 5'-cap structure.

32. The nucleic acid according to claim 31, wherein the 5'-cap structure is selected from m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7GpppN, m7GpppNmpNp, m7GpppNmpNmp; preferably m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

33. A nucleic acid according to any one of the preceding claims, wherein the nucleic acid is delivered by a liver-specific targeted delivery system.

34. The nucleic acid of claim 33, wherein the liver-specific targeted delivery system is lipid nanoparticles or GalNAc delivery technology.

35. A vector or plasmid comprising a nucleic acid according to any one of the preceding claims or a DNA sequence corresponding thereto.

36. A host cell comprising the nucleic acid of any one of claims 1-34 or the vector or plasmid of claim 35.

37. A pharmaceutical composition comprising the nucleic acid of any one of claims 1-34 and a delivery agent.

38. The pharmaceutical composition of claim 37, wherein the delivery agent is selected from the group consisting of liposomes, lipid nanoparticles, lipidoids, polymers, lipoplexes, microvesicles, exosomes, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics, nanotubes, conjugates, and combinations thereof.

39. The pharmaceutical composition of claim 37 or 38, wherein the delivery agent is a lipid nanoparticle.

40. The pharmaceutical composition of any one of claims 37-39, wherein the pharmaceutical composition is formulated as a lipid nanoparticle encapsulating the nucleic acid in a lipid shell.

41. The pharmaceutical composition of any one of claims 37-40, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

42. A nucleic acid according to any one of claims 1 to 34 or a pharmaceutical composition according to any one of claims 37 to 41 for use as a medicament.

43. A nucleic acid according to any one of claims 1-34 or a pharmaceutical composition according to any one of claims 37-41 for use in reducing uric acid levels in a subject.

44. A nucleic acid according to any one of claims 1-34 or a pharmaceutical composition according to any one of claims 37-41 for use in treating or preventing gout, gouty arthritis, hyperuricemia or tumor lysis syndrome in a subject.

45. A method for reducing uric acid levels in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a nucleic acid according to any one of claims 1-34 or a pharmaceutical composition according to any one of claims 37-41.

46. ​​The method of claim 45, wherein the subject is a human or a non-human mammal.

47. The method of claim 45 or 46, wherein the administration is parenteral, or is performed via intramuscular, subcutaneous or intravenous administration.

48. The method of any one of claims 45-47, wherein the administration is one or more times at a frequency of about once a day, about once every two days, about once every three days, about once every four days, about once every five days, about once every six days, about once a week, about once every two weeks, or about once a month.

49. The method of any one of claims 45-48, wherein the lipid nanoparticles encapsulating the nucleic acid are endocytosed by cells in the subject.

50. The method of any one of claims 45-49, wherein the nucleic acid is expressed as urate oxidase, a functional fragment or variant thereof by cells, preferably hepatocytes, in the subject.

51. The method of claim 50, wherein the produced urate oxidase, functional fragment or variant thereof is secreted from hepatocytes into the blood circulation, or is retained in hepatocytes, or both.

52. A method for treating or preventing gout, gouty arthritis, or hyperuricemia in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a nucleic acid according to any one of claims 1-34 or a pharmaceutical composition according to any one of claims 37-41.

53. The method of claim 52, wherein the subject is a human or a non-human mammal.

54. The method of claim 52 or 53, wherein the administration is parenteral, or is performed via intramuscular, subcutaneous or intravenous administration.

55. The method of any one of claims 52-54, wherein the administration is one or more times at a frequency of about once a day, about once every two days, about once every three days, about once every four days, about once every five days, about once every six days, about once a week, about once every two weeks, or about once a month.

56. The method of any one of claims 52-55, wherein the lipid nanoparticles encapsulating the nucleic acid are endocytosed by cells in the subject.

57. The method of any one of claims 52-56, wherein the nucleic acid is expressed by cells in the subject.

58. A method of treating or preventing tumor lysis syndrome in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a nucleic acid according to any one of claims 1-34 or a pharmaceutical composition according to any one of claims 37-41.

59. The method of claim 58, wherein the subject is a human or a non-human mammal.

60. The method of claim 58 or 59, wherein the administration is parenteral, or is performed via intramuscular, subcutaneous, or intravenous administration.

61. The method of any one of claims 58-60, wherein the administration is one or more times at a frequency of about once a day, about once every two days, about once every three days, about once every four days, about once every five days, about once every six days, about once a week, about once every two weeks, or about once a month.

62. The method of any one of claims 58-61, wherein the lipid nanoparticles encapsulating the nucleic acid are endocytosed by cells in the subject.

63. The method of any one of claims 58-62, wherein the nucleic acid is expressed as urate oxidase, a functional fragment or variant thereof by cells, preferably hepatocytes, in the subject.

64. The method of claim 63, wherein the produced urate oxidase, functional fragment or variant thereof is secreted from hepatocytes into the blood circulation, or is retained in hepatocytes, or both.

65. A kit comprising a nucleic acid according to any one of claims 1-34 or a pharmaceutical composition according to any one of claims 37-41 and instructions for use.

66. A method for enhancing the expression of an open reading frame (ORF) encoding a uricase fusion protein in a nucleic acid, wherein the uricase fusion protein is a fusion protein of a signal peptide and uricase, and the signal peptide is fused to the N-terminus of the uricase, the method comprising inserting a coding sequence of a spacer sequence between the coding sequence of the signal peptide and the coding sequence of the uricase in the ORF.

67. A method for enhancing the expression of an open reading frame (ORF) encoding a uricase fusion protein in a nucleic acid, wherein the uricase fusion protein is a fusion protein comprising a signal peptide, an optional spacer sequence, a half-life extension element, an optional linker and uricase from the N-terminus to the C-terminus, or a fusion protein comprising a signal peptide, an optional spacer sequence, uricase, an optional linker and a half-life extension element from the N-terminus to the C-terminus, the method comprising adding a coding sequence of a half-life extension element and a coding sequence of an optional linker to the 5' or 3' end of the coding sequence of uricase in the ORF.

68. The method of claim 66 or 67, wherein the uricase comprises the amino acid sequence of any one of SEQ ID NOs: 1-5 and 9-10, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-5 and 9-10.

69. The method of any one of claims 66-68, wherein the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 22-45, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 22-45.

70. The method of any one of claims 66-69, wherein the spacer sequence comprises the amino acid sequence of any one of SEQ ID NOs: 50-53 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 50-53.

71. The method of any one of claims 67-70, wherein the half-life extension element comprises SEQ ID NO: 381 or 382, ​​or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 381 or 382.

72. The method of any one of claims 67-71, wherein the linker comprises SEQ ID NO: 383 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:

383.

73. The method of any one of claims 66-72, wherein the ORF is codon optimized.

74. The method of any one of claims 66-73, wherein the ORF is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 202-212, 386-389, or their corresponding DNA sequences.

75. The method of any one of claims 66-74, wherein the 5' end of the ORF further comprises a 5'-untranslated region (5'-UTR).

76. The method of claim 75, wherein the 5'-UTR comprises any one of SEQ ID NOs: 54-57, 384, or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 54-57, 384, or a DNA sequence corresponding thereto.

77. A method according to claim 75 or 76, wherein the 5'-UTR comprises SEQ ID NO:55, 56 or 384 or its corresponding DNA sequence or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:55, 56 or 384 or its corresponding DNA sequence.

78. The method of any one of claims 66-77, wherein the 3' end of the ORF further comprises a 3'-untranslated region (3'-UTR).

79. The method of claim 78, wherein the 3'-UTR comprises any one of SEQ ID NOs: 58-62, 385, or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 58-62, 385, or a DNA sequence corresponding thereto.

80. The method of claim 78 or 79, wherein the 3'-UTR comprises SEQ ID NO:59, 61 or 385, or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:59, 61 or 385, or a DNA sequence corresponding thereto.

81. The method of any one of claims 66-80, wherein the nucleic acid obtained comprises, in order from 5' to 3', a 5'-UTR, an ORF encoding a fusion protein comprising a signal peptide, a spacer sequence and uricase from N-terminus to C-terminus, or a fusion protein comprising a signal peptide, a spacer sequence, a half-life extending element, an optional linker and uricase from N-terminus to C-terminus, or a fusion protein comprising a signal peptide, a spacer sequence, a half-life extending element, an optional linker and a half-life extending element from N-terminus to C-terminus, a 3'-UTR, and a 3'-poly(A) sequence, wherein the ORF is selected from any one of SEQ ID NOs: 202-212, 386-389.

82. The method of claim 81, wherein the 5'-UTR is selected from any one of SEQ ID NOs: 54-57, 384, and the 3'-UTR is selected from any one of SEQ ID NOs: 58-62, 385.

83. The method of any one of claims 66-82, wherein the 3' end of the ORF further comprises a 3'-polyadenylation sequence.

84. The method of any one of claims 66-83, wherein the nucleic acid is DNA or mRNA.

85. The method of any one of claims 66-84, wherein the nucleic acid comprises at least one chemical modification.

86. The method of claim 85, wherein the chemical modification is selected from one or more of 5-methylcytidine, 5-methoxyuridine, pseudouridine, N6-methyladenosine, or N1-methylpseudouridine.

87. The method of claim 85 or 86, wherein the chemical modification is N1-methylpseudouridine.

88. The method of any one of claims 66-87, wherein at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or 100% of the uridine in the nucleic acid is chemically modified to N1-methylpseudouridine.

89. The method of any one of claims 66-88, wherein the mRNA further comprises a 5'-cap structure.

90. The method according to claim 89, wherein the 5'-cap structure is selected from m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7GpppN, m7GpppNmpNp, m7GpppNmpNmp; preferably m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

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