Optimized uricase nucleic acid molecules, expression cassettes and uses thereof

Abstract

The present application provides an optimized nucleic acid molecule for expressing a mammalian ancestral uricase, a uricase gene therapy expression cassette, a gene delivery system and uses thereof. The uricase gene therapy expression cassette of the present application is capable of expressing a mammalian ancestral uricase having urate oxidase activity in target cells, preferably hepatocytes, thereby reducing serum uric acid levels, and can be used for preventing or treating diseases that can be ameliorated by reducing serum uric acid levels.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biomedicine, specifically relating to an optimized uricase nucleic acid molecule, a transgenic expression cassette containing the nucleic acid molecule, a gene delivery system, a pharmaceutical composition, and its application in reducing serum uric acid levels and / or treating uric acid-related diseases. Background Technology

[0002] Uric acid is the end product of purine metabolism in the human body, and an imbalance between its production and excretion can lead to elevated serum uric acid levels. Due to the pseudogenotyping of the human uricase gene during evolution, the human body cannot express functional uricase, and therefore cannot further oxidize uric acid into the more water-soluble allantoin, thus making it more prone to uric acid accumulation.

[0003] Elevated serum uric acid levels can cause or worsen various diseases, including hyperuricemia, gout, gouty arthritis, uric acid nephropathy, uric acid kidney stones, chronic kidney disease, and tumor lysis syndrome. In joints and soft tissues, urate crystals can induce NLRP3 inflammasome activation, triggering acute and chronic gout inflammation. In the kidneys, uric acid and urate deposition can lead to renal tubular damage, interstitial inflammation, fibrosis, and stone formation. In tumor lysis syndrome, the release of nucleic acids from massive cell lysis can cause a sharp increase in serum uric acid, inducing acute uric acid nephropathy and acute kidney injury. Furthermore, elevated serum uric acid is also associated with metabolic syndrome, obesity, insulin resistance, type 2 diabetes, hypertension, and cardiovascular disease.

[0004] Current uric acid-lowering treatments mainly include drugs that inhibit uric acid production, drugs that promote uric acid excretion, and recombinant uricase protein preparations. Drugs that inhibit uric acid production require long-term use and pose risks such as hypersensitivity reactions or cardiovascular safety issues. Drugs that promote uric acid excretion are affected by kidney function and may increase the risk of uric acid kidney stones. While recombinant uricase protein preparations can directly break down uric acid, they have high immunogenicity, require repeated administration, and may lead to infusion reactions, allergic reactions, the development of drug-resistant antibodies, and decreased efficacy. Therefore, current treatment methods are insufficient to achieve long-term, stable, and safe control of serum uric acid.

[0005] Gene therapy offers a novel treatment strategy for uric acid-related diseases. By delivering nucleic acid molecules encoding functional uricases into the body, specific tissues can express uricases long-term, sustaining the catalysis of uric acid and thus reducing serum uric acid levels. The liver, with its robust metabolic and protein expression capabilities, is an ideal target tissue for uricase gene therapy. AAV vectors possess the potential for long-term expression and tissue-targeted delivery, making them suitable for liver gene therapy. However, existing uricase gene therapy expression cassettes still face challenges such as insufficient coding sequence expression efficiency, inadequate promoter activity and specificity, limited AAV packaging capacity, risks of expression in non-target tissues, and insufficient long-term efficacy in vivo.

[0006] Therefore, there is still a need in the field to develop an optimized uricase nucleic acid molecule and its expression cassette that can be adapted to gene delivery systems such as AAV and effectively express uricase protein with uricase oxidase activity in target cells, thereby reducing serum uric acid levels and treating or improving hyperuricemia, gout and related diseases. Summary of the Invention

[0007] The purpose of this invention is to provide an optimized uricase nucleic acid molecule that encodes mammalian ancestral uricase, which can efficiently express uricase protein with uricase oxidase activity in mammalian cells.

[0008] Another object of the present invention is to provide a transgenic expression cassette comprising the optimized uricase nucleic acid molecule, which can efficiently express uricase protein with uricase oxidase activity, thereby continuously catalyzing the decomposition of uric acid and reducing serum uric acid levels.

[0009] To achieve the above objectives, the present invention adopts the following technical solution.

[0010] In a first aspect, the present invention provides a nucleic acid molecule that encodes a mammalian ancestral uricase with an amino acid sequence as shown in SEQ ID NO: 12, wherein the nucleotide sequence of the nucleic acid molecule has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] In some preferred embodiments, the nucleotide sequence of the nucleic acid molecule has at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] In some preferred embodiments, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] In some preferred embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0014] In a second aspect, the present invention provides a transgenic expression cassette comprising: a promoter, a nucleic acid molecule as described in the first aspect of the present invention, and PolyA.

[0015] In some preferred embodiments, the promoter is a liver-specific promoter.

[0016] In some preferred embodiments, the promoter is selected from the HCR-hAAT promoter, the FA promoter shown in SEQ ID NO: 5, and the FA1 promoter shown in SEQ ID NO: 6.

[0017] In some preferred embodiments, PolyA is bGH polyA.

[0018] In some implementations, the transgene expression cassette also includes two ITRs located at both ends.

[0019] In some implementations, the two ITRs are either normal ITRs or shortened ITRs, each independently.

[0020] In some implementations, the ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVDJ, poultry AAV, cattle AAV, dog AAV, horse AAV, sheep AAV, goat AAV, or mouse AAV ITR.

[0021] In some embodiments, the transgenic expression cassette also includes an intron located between the promoter and the nucleic acid molecule. In some preferred embodiments, the intron is SV40.

[0022] In some implementations, the transgenic expression cassette also includes a Kozak sequence located between the promoter and the nucleic acid molecule.

[0023] In some implementations, the transgenic expression cassette also includes a WPRE sequence located between the nucleic acid molecule and PolyA.

[0024] In some preferred embodiments, the nucleotide sequence of the transgenic expression cassette is shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

[0025] In a third aspect, the present invention provides a gene delivery system comprising the transgenic expression cassette and AAV capsid protein described in the second aspect of the present invention.

[0026] In some embodiments, the AAV capsid protein is selected from AAV1, AAV3, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ8, AAV-LK03, Anc80L65, AAVrh39, AAVrh43, AAVrh74, AAV2G9, AAV.PHP, AAV3B, and AAV843 capsid proteins.

[0027] In some preferred embodiments, the AAV capsid protein is the AAV843 capsid protein (see, for example, CN114457086B for the AAV843 capsid protein).

[0028] In a fourth aspect, the present invention provides the use of the nucleic acid molecule described in the first aspect of the present invention, the transgenic expression cassette described in the second aspect of the present invention, or the gene delivery system described in the third aspect of the present invention in the preparation of a medicament for treating a disease.

[0029] In some embodiments, the disease is one that can be improved by lowering serum uric acid levels.

[0030] In some embodiments, the disease is selected from hyperuricemia, gout, gouty arthritis, uric acid nephropathy, uric acid kidney stones, chronic kidney disease, tumor lysis syndrome, and metabolic diseases caused or aggravated by elevated serum uric acid.

[0031] In a fifth aspect, the present invention provides a medicine comprising: a nucleic acid molecule as described in the first aspect of the present invention, a transgenic expression cassette as described in the second aspect of the present invention, or a gene delivery system as described in the third aspect of the present invention; and optionally an excipient.

[0032] In some embodiments, the excipient is a pharmaceutically acceptable carrier, diluent, buffer, stabilizer, osmotic pressure regulator, surfactant, preservative, cryoprotectant, or any combination thereof.

[0033] In some implementations, the drug is an injectable form.

[0034] In some implementations, the drug is suitable for systemic or local injection.

[0035] In some implementations, the drug is administered via intravenous injection, portal vein injection, hepatic artery injection, intraperitoneal injection, subcutaneous injection, or intramuscular injection.

[0036] In some preferred embodiments, the drug is administered via intravenous injection.

[0037] In a sixth aspect, the present invention provides a method for treating a disease that can be improved by lowering serum uric acid levels, comprising administering a therapeutically effective amount of the drug described in the fifth aspect of the present invention to a subject in need.

[0038] In some implementations, the subjects are non-human animals. In some implementations, the subjects are mice.

[0039] In some embodiments, the subject is a mammal. In some preferred embodiments, the subject is a human.

[0040] In a seventh aspect, the present invention provides a method for delivering a nucleic acid molecule encoding a mammalian ancestral uricase to target cells, the amino acid sequence of which is shown in SEQ ID NO: 12, the method comprising:

[0041] 1) The transgenic expression cassette described in the second aspect of the present invention is packaged in AAV capsid protein to form the gene delivery system described in the third aspect of the present invention; and

[0042] 2) Contact the target cells with the gene delivery system.

[0043] In some embodiments, the target cell is a mammalian cell.

[0044] In some embodiments, the target cells are hepatocytes.

[0045] In some embodiments, the target cell is an isolated cell.

[0046] In some embodiments, the contact is performed under in vitro, ex vivo, or in vivo conditions.

[0047] Compared with the prior art, the present invention has at least the following beneficial effects:

[0048] 1. The nucleic acid molecules of the present invention can express uricase protein with uricase oxidase activity at a high expression level in host cells.

[0049] 2. The transgenic expression cassette of the present invention is compatible with the AAV delivery system, enabling efficient expression of uricase in hepatocytes. The gene delivery system of the present invention can deliver the uricase expression cassette to target tissues in vivo, allowing subjects to continuously express uricase, thereby reducing serum uric acid levels and decreasing the need for repeated dosing.

[0050] 3. This invention improves uricase expression levels and tissue targeting by optimizing the coding sequence, promoter, and expression cassette structure, making it suitable for developing long-acting gene therapy drugs that lower uric acid.

[0051] The foregoing and other aspects of this disclosure are set forth in more detail in the following description. Attached Figure Description

[0052] Figure 1 The diagram shows the construction of AUX optimization 1, AUX optimization 2, AUX optimization 3, and AUX optimization 4.

[0053] Figure 2 A schematic diagram of the construction of promoters FA, FA1, FA2 and FA3 is shown.

[0054] Figure 3A bar chart showing the activity comparison of different liver-specific promoters driving Gluc expression in Huh7 cells is presented. The FA promoter is compared with the negative control (Ctrl), LP1, HLP, AAT, Lxp2.1, TTRmut, and HCR-hAAT. <0.05, <0.01, <0.001; Compared with other promoters, FA1 has #p<0.05, ##p<0.01, and ###p<0.001.

[0055] Figure 4 A bar chart showing the activity comparison of different liver-specific promoters driving Gluc expression in Huh7 cells is presented. The FA promoter is compared with the negative control (Ctrl), AAT, FA2, and FA3. <0.05, <0.01, <0.001; Compared with other promoters, FA1 has #p<0.05, ##p<0.01, and ###p<0.001.

[0056] Figure 5 The dynamic changes in Gluc expression in mouse serum mediated by different promoters are shown.

[0057] Figure 6 The bar chart shows tissue-specific analysis of Gluc mRNA expression driven by different promoters in various mouse tissues.

[0058] Figure 7 A schematic diagram of the construction of expression boxes B351, B352, B353, B355, and B356 is shown.

[0059] Figure 8 The table shows the uricase activity per unit volume of crude enzyme solutions extracted after transfection of Huh7 cells with expression cassettes B351, B352, B353, B355, and B356. Compared with B351, NC, B355, and B356... <0.05, <0.01, <0.001; Comparing NC, B351, B355, B356 with B352, #p<0.05, ##p<0.01, ###p<0.001; Comparing NC, B351, B352, B355, B356 with B353, &p<0.05, &&p<0.01, &&&p<0.001.

[0060] Figure 9The image shows the crude enzyme solution extracted after transfection of Huh7 cells with B351, B352, and B353 expression cassettes, stained with Coomassie Brilliant Blue after SDS-PAGE.

[0061] Figure 10 The changes in serum uric acid levels in mice after B351, B352, and B353 expression cassettes were packaged into the AAV843 viral vector and injected into a mouse model of hyperuricemia were shown.

[0062] Figure 11 The changes in liver uricase activity in mice after B351, B352, and B353 expression cassettes were packaged into the AAV843 viral vector and injected into a mouse model of hyperuricemia were shown. <0.05, <0.01, <0.001.

[0063] Figure 12 A flowchart illustrating the construction of a mouse model of acute gout is shown.

[0064] Figure 13 The changes in paw swelling over time are shown in mice with an acute gout model. The B351+MSU, B352+MSU, and B353+MSU groups are compared with the UOX-KO+MSU group. Detailed Implementation

[0065] 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 to which this invention pertains. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0066] Unless otherwise stated, the nucleic acid or polynucleotide sequences listed in this article are in single-stranded form, oriented from 5' to 3', from left to right. The nucleotides and amino acids provided in this article follow the format recommended by the IUPACIUB Biochemical Nomenclature Committee, with amino acids using either single-letter or three-letter codes.

[0067] the term

[0068] In this document, the term "polynucleotide" is a synonym for "nucleic acid" and refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, their mixed sequences, or similar. Polynucleotides may include modified nucleotides, such as methylated or restricted nucleotides and nucleotide analogs.

[0069] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0070] In this paper, the term "codon optimization" refers to a polynucleotide sequence modified from its natural form. Such modifications result in differences in one or more base pairs, with or without alteration in the corresponding amino acid sequence, which may enhance or suppress gene expression and / or cellular responses to the modified polynucleotide sequence.

[0071] In this document, the terms “patient” and “subject” are used interchangeably and in their conventional sense, referring to an organism that suffers from or is susceptible to a condition that can be prevented or treated by administration of the medicament of the present invention, and including humans and non-human animals (e.g., rodents or other mammals).

[0072] In this document, the term “treatment” includes: (1) suppressing a symptom, disease or condition, i.e., preventing, reducing or delaying the development of a disease or its recurrence or the development of at least one of its clinical or subclinical symptoms; or (2) alleviating a disease, i.e. causing the disappearance or elimination of at least one of a symptom, disease or condition or its clinical or subclinical symptoms.

[0073] In this article, the term "therapeutic effective dose" or "effective dose" refers to the dose that produces the therapeutic effect to be achieved when it is administered.

[0074] In this article, the term “improvement” refers to the improvement of symptoms related to the disease, and may refer to the improvement of at least one parameter that measures or quantifies the symptom.

[0075] In this document, the term “prevention” of a symptom, disease, or condition includes: preventing, delaying, or reducing the incidence and / or likelihood of developing at least one clinical or subclinical symptom of a symptom, disease, or condition in a subject who may have or be susceptible to the symptom, disease, or condition but has not yet experienced or exhibited clinical or subclinical symptoms of the symptom, disease, or condition.

[0076] In this article, the terms "local application" or "local route" refer to administration of drugs that have a local effect.

[0077] In this paper, the terms “transduction,” “transfection,” and “conversion” refer to the process of delivering exogenous nucleic acids into host cells, followed by transcription and translation of the polynucleotide product, which includes the introduction of exogenous polynucleotides into host cells using recombinant viruses.

[0078] In this paper, the term "gene expression" or "expression" refers to the process by which gene transcription, translation, and post-translational modification produce the RNA or protein product of the gene.

[0079] In this paper, the term "targeting" refers to the preferential entry of a vector into certain cells or tissues, whereby the viral genome or the sequence carried by the recombinant transgene is further expressed.

[0080] In this paper, the term "vector" refers to one or more macromolecules that encapsulate polynucleotides, facilitating their delivery to target cells in vitro or in vivo. Types of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene or non-gene delivery vectors.

[0081] In this article, the terms “expression cassette,” “transgenic cassette,” and “transgenic expression cassette” are used interchangeably to refer to a polynucleotide fragment encoding a specific protein, polypeptide, or RNAi element that can be cloned into a plasmid vector or packaged into a viral particle (such as AAV) to deliver the transgenic product to target cells.

[0082] In this document, the terms “optional” or “optional” mean that the events or circumstances described below may occur but are not required to occur.

[0083] In this paper, the term "inverted terminal repeat (ITR)" includes any AAV viral terminal repeat or synthetic sequence that forms a hairpin structure and is used as a cis element to mediate viral replication, packaging, and integration.

[0084] 1. Nucleic acid molecules encoding uricase, an ancestral enzyme in mammals.

[0085] In some embodiments, the nucleic acid molecule is AUX Optimized 1 (SEQ ID NO: 1).

[0086] In some embodiments, the nucleic acid molecule is AUX optimized 2 (SEQ ID NO: 2).

[0087] AUX Optimization 1 and AUX Optimization 2 encode mammalian ancestral uricase An19 / 22 and have undergone sequence optimization suitable for mammalian cell expression. In some embodiments, AUX Optimization 1 or AUX Optimization 2 includes one or more sequence elements capable of improving transcription, mRNA processing, mRNA stability, translation efficiency, or protein expression levels.

[0088] In some embodiments, the nucleic acid molecule may express a uricase protein with uricase oxidase activity in a host cell. The host cell is preferably a mammalian cell, more preferably a hepatocyte or liver-derived cell.

[0089] 2. Transgenic expression cassette

[0090] The transgenic expression cassette of the present invention includes: a promoter, a nucleic acid molecule encoding uricase, a mammalian ancestor, and PolyA.

[0091] In some preferred embodiments, the transgenic expression cassette includes an FA promoter or FA1 promoter, an optional SV40 intron, a Kozak sequence, a nucleic acid molecule encoding a mammalian ancestral uricase, an optional WPRE sequence, and bGH polyA.

[0092] In some preferred embodiments, the transgenic expression cassette includes an FA1 promoter, an optional SV40 intron, an AUX-optimized 1 nucleic acid molecule encoding a mammalian ancestral uricase, an optional WPRE sequence, and bGH polyA.

[0093] In some preferred embodiments, the transgenic expression cassette includes an FA1 promoter, an optional SV40 intron, an AUX-optimized 2 nucleic acid molecule encoding a mammalian ancestral uricase, an optional WPRE sequence, and bGH polyA.

[0094] In some embodiments, the transgenic expression cassette is the B351 expression cassette. In some embodiments, the transgenic expression cassette is the B352 expression cassette. In some embodiments, the transgenic expression cassette is the B353 expression cassette.

[0095] 3. Gene delivery systems

[0096] The gene delivery system of the present invention includes a transgene expression cassette and an AAV capsid protein.

[0097] In some implementations, the AAV capsid protein may be derived from natural AAV serotypes, artificially modified AAV capsids, chimeric AAV capsids, mutant AAV capsids, directed evolution AAV capsids, or variants thereof.

[0098] In some implementations, the gene delivery system is capable of delivering nucleic acid molecules encoding uricase, a mammalian ancestor, into target cells.

[0099] In some implementations, the target cells are mammalian cells.

[0100] In some preferred embodiments, the target cells are hepatocytes.

[0101] In some implementations, the gene delivery system can express uricase protein with uricase oxidase activity in the liver of the subject, thereby reducing the subject's serum uric acid level.

[0102] In some preferred embodiments, the gene delivery system comprises a B351 expression cassette and an AAV843 capsid protein, with nucleotide sequences as shown in SEQ ID NO: 9. In some preferred embodiments, the gene delivery system comprises a B352 expression cassette and an AAV843 capsid protein, with nucleotide sequences as shown in SEQ ID NO: 10. In some preferred embodiments, the gene delivery system comprises a B353 expression cassette and an AAV843 capsid protein, with nucleotide sequences as shown in SEQ ID NO: 11.

[0103] 4. Pharmaceutical Uses

[0104] The nucleic acid molecule encoding uricase, a transgenic expression cassette containing the nucleic acid molecule, a gene delivery system containing the transgenic expression cassette, and a drug or pharmaceutical composition containing the present invention can be used to lower serum uric acid levels, thereby being used to treat diseases that can be improved by lowering serum uric acid levels, such as hyperuricemia, gout, gouty arthritis, uric acid nephropathy, uric acid kidney stones, chronic kidney disease, tumor lysis syndrome, and metabolic diseases caused or aggravated by elevated serum uric acid.

[0105] In some embodiments, the medicine of the present invention is used to prevent, treat, alleviate, improve, or delay diseases that can be improved by lowering serum uric acid levels.

[0106] In some embodiments, the drug of the present invention lowers serum uric acid levels by expressing uricase. In some embodiments, the uricase is a mammalian ancestral uricase. In some embodiments, the amino acid sequence of the uricase is as shown in SEQ ID NO: 12.

[0107] In some embodiments, the drug of the present invention converts uric acid in the subject's body into allantoin or uric acid oxidation metabolites, which are more water-soluble.

[0108] In some embodiments, the subjects are non-human animals (e.g., chimpanzees and other ape and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domesticated mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including poultry, wild birds, and game birds such as chickens, turkeys, and other chickens, ducks, geese, etc.). In some embodiments, the subjects are mammals. In some embodiments, the subjects are humans.

[0109] In some embodiments, the medicament of the present invention is used to lower serum uric acid levels, reduce urate crystal deposition, improve urate crystal-induced inflammatory responses, improve renal uric acid deposition or crystal formation, reduce the risk of uric acid kidney injury, reduce the risk of uric acid kidney stones, and / or improve metabolic abnormalities caused or aggravated by elevated serum uric acid.

[0110] In some implementations, the drug may be delivered in single or multiple doses.

[0111] The effective amount of a drug can vary depending on the mode of administration and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the drug, such as tissue distribution, bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0112] In some implementations, more than one dose (e.g., two, three, four or more doses) can be used to achieve the desired level of protein expression at various time intervals (e.g., daily, weekly, monthly, yearly, etc.).

[0113] In some embodiments, the drug may be present in single-dose or multi-dose forms, such as in ampoules, vials, pre-filled syringes, lyophilized formulation containers, or other containers suitable for storage and administration. In some embodiments, the drug may be stored in a liquid, frozen, or lyophilized state. In some embodiments, the lyophilized formulation may be reconstituted with sterile water for injection, physiological saline, buffer solution, or other pharmaceutically acceptable solvent before use.

[0114] 5. Treatment methods

[0115] The present invention also provides a method for treating, preventing, alleviating or improving a disease, comprising administering a therapeutically effective amount of the drug, transgenic expression cassette or gene delivery system of the present invention to a subject in need.

[0116] In some embodiments, the method includes administering a therapeutically effective amount of a drug containing a nucleic acid molecule encoding a mammalian ancestral uricase to a subject in need, thereby causing the subject to express a uricase protein with uricase oxidase activity, thereby reducing the subject's serum uric acid level.

[0117] In some embodiments, the method is used to treat, prevent, alleviate, or improve diseases that can be improved by lowering serum uric acid levels. In some embodiments, the diseases include, but are not limited to, hyperuricemia, gout, gouty arthritis, uric acid nephropathy, uric acid kidney stones, chronic kidney disease, tumor lysis syndrome, and metabolic diseases caused or aggravated by elevated serum uric acid.

[0118] In some embodiments, the treatment method further includes detecting the subject's serum uric acid level, uric acid oxidation metabolite level, liver function indicators, kidney function indicators, inflammatory indicators, immune response indicators, anti-AAV antibody level, anti-uricase antibody level, and / or target protein expression level before, during, and / or after administration.

[0119] In some embodiments, when the method is used to treat hyperuricemia or gout, the method further includes detecting the subject's serum uric acid level, urate crystal deposition, joint swelling, pain level, inflammatory cell infiltration, renal uric acid deposition, renal function indicators, and / or gout attack frequency.

[0120] In some embodiments, the treatment methods may be used alone or in combination with other treatment methods. In some embodiments, the other treatment methods include drugs that inhibit uric acid production, drugs that promote uric acid excretion, anti-inflammatory drugs, analgesics, immunomodulatory drugs, urine alkalizing drugs, nephroprotective drugs, or dietary control.

[0121] In some embodiments, the method reduces the subject's serum uric acid level. In some embodiments, the method reduces the subject's serum uric acid level to a normal range. In some embodiments, the method reduces urate crystal deposition, alleviates gouty inflammation, reduces the frequency of gout attacks, improves uric acid-induced kidney damage, reduces the risk of uric acid kidney stones, and / or improves metabolic abnormalities caused or exacerbated by elevated serum uric acid.

[0122] The present disclosure will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the embodiments that do not specify specific conditions are performed under conventional conditions known in the art or under conditions recommended by the manufacturer. Example

[0123] Example 1: Design and construction of an optimized nucleic acid sequence encoding uricase, a mammalian ancestor

[0124] First, based on the literature (Kratzer JT et al. Evolutionary history and metabolic insights of ancient mammalian uricases[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(10): 3763-3768.), the amino acid sequence An19 / 22 (SEQ ID NO: 12) of the mammalian ancestor uricase was obtained, hereinafter referred to as AUX. According to the correspondence rules between amino acids and codons, the AUX protein sequence was converted into a DNA sequence, and human codon optimization was performed to obtain the codon-optimized AUX DNA sequences: AUX Optimization 1 (SEQ ID NO: 1), AUX Optimization 3 (SEQ ID NO: 3), and AUX Optimization 4 (SEQ ID NO: 4).

[0125] Furthermore, inserting one chimeric intron (rGlobin intron) into the sequence of AUX optimization 1 yields the sequence: AUX optimization 2 (SEQ ID NO: 2).

[0126] The construction diagrams for AUX optimization 1, AUX optimization 2, AUX optimization 3, and AUX optimization 4 are shown below. Figure 1 As shown.

[0127] Example 2: Design and Construction of Novel Liver-Specific Promoters

[0128] 2.1 Design of liver-specific promoter sequences

[0129] To obtain a short promoter fragment with high activity, high liver specificity, and favorable for adapting to the packaging capacity of recombinant AAV vectors, this embodiment tandemly connects two liver-specific promoters—the FIX promoter and the AAT promoter—and further inserts them into sites that bind to liver-specific transcription factors, such as... Figure 2 As shown. The specific construction method is as follows:

[0130] The FIX promoter sequence and AAT promoter sequence were obtained from the National Center for Biotechnology Information (NCBI). The -219 to -1 region of the FIX promoter (i.e., the core region of the FIX promoter, SEQ ID NO: 13) and the -261 to +44 region of the AAT promoter (i.e., the core region of the AAT promoter, SEQ ID NO: 14) were selected. The selected regions of the FIX promoter and AAT promoter were tandemly connected using the restriction endonuclease XbaI recognition sequence (TCTAGA) as the linker sequence to obtain the FA promoter (SEQ ID NO: 5).

[0131] Furthermore, based on the FA promoter, a portion of the sequence in the core region of the AAT promoter (i.e., the -203 to -191, -188 to -174, -158 to -144 and / or -16 to -9 region sequences of the AAT promoter) is replaced with liver-enriched transcription factor binding sites (i.e., HNF4α binding site (CTGACCTTTGCCC), HNF1 binding site (TGGTTAATTTTTAAA), CEBPα binding site (GATTCTGATTACAAA) and / or HNF3 binding site (CTGTTTAC)) to obtain the FA1 (SEQ ID NO: 6), FA2 (SEQ ID NO: 7) and FA3 (SEQ ID NO: 8) promoters.

[0132] 2.2 Construction of Gluc fluorescence screening system vector plasmid

[0133] To evaluate the transcriptional activity of promoters, a Gluc fluorescence screening system vector plasmid was constructed. The FA, FA1, FA2, or FA3 promoters were ligated upstream of the Gluc reporter gene, resulting in the pscAAV-FA-Gluc, pscAAV-FA1-Gluc, pscAAV-FA2-Gluc, and pscAAV-FA3-Gluc vector plasmids. Gluc is a Gaussian luciferase, a protein that, after intracellular expression, is secreted extracellularly and catalyzes the oxidation of the substrate coelenterate to produce a chemiluminescent signal. The expression level of Gluc can be quantitatively detected by measuring the luminescence intensity after the reaction of culture medium or serum samples with coelenterate, thereby evaluating and comparing the transcriptional activity of different promoters.

[0134] The target gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd. During gene synthesis, KpnI and HpaI restriction endonuclease sites were introduced at the 5' and 3' ends of the target sequence, respectively. The synthesized target gene was double-digested with KpnI and HpaI, and then ligated to the plasmid backbone pscAAV-CB-Gluc, which had been linearized with the same restriction endonuclease, using T4 DNA ligase. The ligation product was transformed into *E. coli* Top10 competent cells, plated, and single colonies were picked for sequencing identification. After sequencing confirmation, a large number of plasmids were extracted for subsequent functional verification experiments.

[0135] Meanwhile, several known liver-specific promoters were selected as controls, including the HCR-hAAT promoter (SEQ ID NO: 15), HLP promoter (SEQ ID NO: 16), LP1 promoter (SEQ ID NO: 17), TTRmut promoter (SEQ ID NO: 18), Lxp2.1 promoter (SEQ ID NO: 19), and AAT promoter. The control promoter-Gluc vector was constructed using the same method as above.

[0136] Example 3: Verification of the promoter's transcriptional activity in cells

[0137] 3.1 Recombinant AAV Reporter Virus Packaging

[0138] The AAV Rep and Cap protein expression plasmid pAAV843-Rep / Cap (AAV843 capsid can be found in CN114457086B for example), the helper plasmid pAdHelper, and the Gluc expression plasmid constructed in Example 2 were mixed in a molar ratio of 1:2:1 and then co-transfected into HEK293 cells using the PEI method. 72 hours after transfection, cells and culture supernatant were collected, and recombinant AAV virus was purified by density gradient centrifugation with iodixanol to obtain Gluc expression viruses AAV843-FA-Gluc, AAV843-FA1-Gluc, AAV843-FA2-Gluc, AAV843-FA3-Gluc, as well as AAV843-HCR-hAAT-Gluc, AAV843-HLP-Gluc, AAV843-LP1-Gluc, AAV843-TTRmut-Gluc, AAV843-Lxp2.1-Gluc, and AAV843-AAT-Gluc.

[0139] 3.2 Huh7 cell infection and Gluc detection

[0140] Huh7 cells (a human hepatocellular carcinoma cell line) were seeded in 12-well plates at a density of 2E5 cells / well and cultured at 37°C in a CO2 incubator until the cell density reached 80%-90%. The cells were then infected with the aforementioned Gluc expression virus at a multiplicity of infection (MOI) of 1 × 10⁻⁶. 5 Forty-eight hours after infection, 20 μL of cell culture supernatant was collected and reacted with coelenterate diluted 1:1000, and the fluorescence intensity was measured using an ELISA reader.

[0141] The results are as follows Figure 3 As shown, after Huh7 cells were infected with different recombinant AAVs, each promoter could drive different levels of Gluc expression. Among them, HCR-hAAT, as a widely used and highly efficient liver-specific promoter in the current technology, had the highest Gluc activity among the control promoters, which was (5.5±0.4)×10. 5 The activity range of the other control promoters was 1×10⁻⁶. 5 Up to 4.2×10 5 .

[0142] The FA promoter-driven gluc activity was (6.36±0.43)×10⁻⁶. 5 It is higher than the other control promoters ( <0.05, <0.001), a 15.6% improvement compared to HCR-hAAT; FA1 promoter-driven Gluc activity was (7.84±0.28)×10 5It is the highest among all tested promoters, with an improvement of 42.5% over HCR-hAAT (###p<0.001) and an improvement of 23.2% over FA (###p<0.001).

[0143] For example Figure 4 As shown, the Gluc activities driven by the modified FA, FA1, FA2 and FA3 promoters were all higher than those of the negative control group (Ctrl) and the AAT promoter; the Gluc activity driven by the FA1 promoter was the highest among all promoters (###p<0.001).

[0144] The above results show that the activity of the heterozygous promoters constructed by FIX and AAT was significantly enhanced. Among them, the FA promoter and FA1 promoter have efficient transcriptional driving ability in human hepatocellular carcinoma cells Huh7, and their activity is significantly better than that of a variety of liver-specific promoters in the prior art. Among them, the transcriptional activity of the FA1 promoter is the highest.

[0145] Example 4: Verification of the promoter's transcriptional activity in mice

[0146] With 5×10 12 Eight-week-old male C57 mice were injected intravenously via the tail vein with the above-mentioned recombinant AAV virus at a dose of vg / kg body weight (n=4 mice / group), while the negative control group (Ctrl) was injected with an equal volume of physiological saline. Blood samples were collected from the orbital sinus before injection and at 1, 2, 3, and 4 weeks after injection. The whole blood was allowed to stand for 1 hour and then centrifuged at 8000 rpm to extract serum. 5 μL of serum was reacted with coelenterate diluted 1:1000, and the chemiluminescence intensity was measured using an ELISA reader.

[0147] The results are as follows Figure 5 As shown, after AAV injection, the serum Gluc activity of mice in each group increased significantly. At week 4, the Gluc activity of mice in the HCR-hAAT group was the highest among the control promoters, at 3242.8±296.3, while the activities of other control groups were in the range of 700 to 2700.

[0148] At week 4, serum Gluc activity in FA group mice was 3515.6±146.3, similar to the HCR-hAAT group (p>0.05), and significantly higher than all other control promoter groups (p<0.05). At week 4, serum Gluc activity in FA1 mice was 4448.4±284.9, significantly higher than all tested promoters (p<0.001), increasing by 37.2% compared to the HCR-hAAT group and by 26.5% compared to the FA group.

[0149] The results of this embodiment show that the FA and FA1 promoters can efficiently drive the expression of target genes in mice. The transcriptional activity of the FA1 promoter in mice is significantly better than that of various liver-specific promoters in the prior art. The transcriptional activity of the FA promoter in mice is higher than that of promoters such as AAT, Lxp2.1, LP1, TTRmut, and HLP. Moreover, with a shorter sequence length, it reaches the transcriptional activity level of the HCR-hAAT promoter.

[0150] Example 5: Tissue-specific verification of promoters

[0151] Based on Example 4, it was confirmed that the FA and FA1 promoters have the ability to enhance the expression of the target gene in mice. Furthermore, the liver specificity of the promoters was verified by quantitative analysis of the target gene mRNA in various mouse tissues.

[0152] With 5×10 12 Eight-week-old male C57 mice were injected intravenously via the tail vein with doses of AAV843-FA-Gluc, AAV843-FA1-Gluc, and AAV843-AAT-Gluc (n=5 mice / group). The negative control group (Ctrl) received only the same volume of saline. Four weeks after injection, the mice were euthanized, and their heart, liver, spleen, lungs, kidneys, brain, quadriceps femoris muscle, and stomach were harvested.

[0153] 20 mg of each tissue sample was collected and processed using an automated RNA extraction kit (Novizan). RNA was then extracted from each tissue using an automated nucleic acid extractor (Novizan, VNP-32P). The RNA was reverse transcribed into cDNA using a reverse transcription kit (Novizan, R433-01). A standard curve was plotted using serially diluted AAV-FA1-Gluc virus of known titers as a standard by real-time quantitative PCR, and absolute quantification of Gluc mRNA in each sample was performed. The tissue specificity of the promoter was analyzed by comparing the GlucmRNA content in each sample.

[0154] The results are as follows Figure 6 As shown, the number of Gluc mRNA copies in the liver was significantly increased in the AAT, FA, and FA1 groups. The number of Gluc mRNA copies in the liver of the FA group was (1.1±0.12)×10⁻⁶. 6 The number of copies / μg RNA was significantly higher than that in the AAT group ((6.6±1.7)×10⁻⁶). 5 copies / μg RNA <0.001), a 61.3% increase compared to the AAT group; the number of Gluc mRNA copies in the liver of the FA1 group was further increased to (1.8±0.18)×10. 6The number of copies / μg RNA was significantly higher than that of the FA group and the AAT group (###p<0.001), with a 68.7% increase relative to the FA group and a 172.1% increase relative to the AAT group.

[0155] In other tissues, there was no significant difference in Gluc mRNA copy number between the FA and FA1 groups and the control group (p>0.05). However, in the heart, the Gluc mRNA copy number in the AAT group was significantly higher than that in the Ctrl group, indicating off-target expression.

[0156] The results of this embodiment show that the FA and FA1 promoters have significant target tissue expression advantages, and the reporter gene expression levels driven in the liver are significantly higher than those driven by the AAT promoter, with FA1 exhibiting the highest transcriptional activity. The FA and FA1 promoters have strict tissue specificity, driving high levels of specific expression only in the liver, with no significant off-target expression in non-target tissues such as the heart, spleen, lungs, and kidneys.

[0157] Example 6: Construction of expression boxes B351, B352, B353, B355 and B356

[0158] Based on the optimized AUX sequence obtained in Example 1, a uricase transgenic expression cassette was further constructed.

[0159] First, using the liver-specific promoter FA1, expression cassette B351 (SEQ ID NO: 9) based on the AUX optimized 1 sequence and expression cassette B352 (SEQ ID NO: 10) based on the AUX optimized 2 sequence were constructed.

[0160] Next, based on the B352 expression cassette, a WPRE sequence was added between the AUX optimization 2 sequence and the PolyA sequence to enhance mRNA stability and / or increase post-transcriptional expression levels, thus constructing the B353 expression cassette (SEQ ID NO: 11).

[0161] Furthermore, based on the B351 expression box, the AUX optimization 1 sequence was replaced with the AUX optimization 3 sequence to construct the B355 expression box; and the AUX optimization 1 sequence was replaced with the AUX optimization 4 sequence to construct the B356 expression box.

[0162] The construction diagrams of expression boxes B351, B352, B353, B355, and B356 are shown below. Figure 7 As shown.

[0163] Example 7: Uricase protein expression of expression cassettes B351, B352, B353, B355 and B356

[0164] Huh7 cells in good growth condition were seeded in 60 mm culture dishes at a density of 1E6 cells / dish. After 24 hours, when the cell confluence reached 80%-90%, the plasmid vectors corresponding to the B351, B352, B353, B355, and B356 expression cassettes were transfected into the cells using PEI transfection reagent. 3 μg of plasmid was transfected into each well, and 2 volumes of PEI reagent were added proportionally. The medium was changed 6 hours after transfection, and the cells were cultured for 72 hours.

[0165] Based on the reference (Duan Y et al. Expression, localization and metabolic function of "resurrected" human urate oxidase in human hepatocytes [J]. International Journal of Biological Macromolecules, 2021, 175: 30-39.), the extraction method of uricase protein was determined as follows: The collected cells were lysed using Tris-HCl buffer (0.1 M, pH 8.7) containing 2% sodium deoxycholate. The cells were centrifuged at 10,000 g for 30 minutes at 4°C. The resulting precipitate was then dissolved in an equal volume of carbonate buffer (0.1 M, pH 10.3) to obtain a crude purified uricase solution.

[0166] Take the same volume of crude enzyme solution for SDS-PAGE electrophoresis and Coomassie brilliant blue staining to detect the AUX protein expression level.

[0167] Take the same volume of crude enzyme solution and react it with 1 mM uric acid solution dissolved in 0.1 M sodium borate buffer (pH 8.5). Continuously monitor the change in absorbance of the reaction solution at a wavelength of 293 nm. Convert the measured rate of absorbance decrease into the substrate consumption rate using Lambert-Beer law, and then calculate the enzyme activity per unit volume of crude enzyme solution.

[0168] The results of the uricase enzyme activity test showed that ( Figure 8 Compared with the NC group, the uricase activity per unit volume of crude enzyme solution in the B351, B352, and B353 groups was significantly increased. The enzyme activity of each transfection group showed an increasing relationship, with the uricase activity per unit volume of the B353 transfection group being significantly better than that of other vector groups.

[0169] Coomassie Brilliant Blue results showed ( Figure 9 Compared with the blank control group (NC), the protein expression levels of crude enzyme solutions in groups B351, B352, and B353 were significantly increased, and the protein expression levels of each transfection group showed an increasing relationship. Among them, the AUX protein expression level in the B353 transfection group was significantly better than that in other vector groups.

[0170] The results show that all vectors can successfully express AUX protein in the hepatocellular carcinoma cell line Huh7, with B353 showing the highest expression efficiency in Huh7.

[0171] Example 8: The therapeutic effects of AAV843-B351, AAV843-B352, and AAV843-B353 on hyperuricemia

[0172] To verify the therapeutic effects of the B351, B352, and B353 expression cassettes on hyperuricemia, the expression cassettes were packaged into a liver-targeting AAV843 viral vector (see, for example, CN114457086B). The viral vector was administered via tail vein injection to 8-week-old male hyperuricemia model mice (purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd., with the uricase gene knocked out in C57 mice) at a dose of 5E12 vg / kg (viral genome number / kg body weight). A hyperuricemia model control group (UOX-KO) and a wild-type control group (WT) (n=5) were also established, receiving only the same volume of physiological saline. Twelve weeks after treatment, all mice were euthanized, and blood and liver tissue samples were collected to detect serum uric acid and hepatic uricase activity. The methods for extracting and detecting hepatic uricase activity were the same as in Example 7.

[0173] Serum uric acid test results as follows Figure 10 As shown, before drug administration, the serum uric acid levels in mice treated with the B351, B352, and B353 vectors (hereinafter referred to as the B351, B352, and B353 groups) were significantly higher than those in the WT group (p<0.001), and there was no significant difference compared with UOX-KO mice (p>0.05). At week 4 after drug administration, the serum uric acid levels in all three vector groups were significantly lower than those in UOX-KO mice, reaching the levels of the WT group.

[0174] Liver uricase activity results as follows Figure 11 As shown, after drug administration, the liver enzyme activity of mice in each group was significantly increased compared with that of UOX-KO mice (p<0.001).

[0175] In summary, the B351, B352, and B353 vectors can significantly increase the expression of uricase in the liver of hyperuricemia model mice, thereby reducing serum uric acid concentration and restoring it to the level of WT mice.

[0176] Example 9: The therapeutic effects of AAV843-B351, AAV843-B352, and AAV843-B353 on gout

[0177] Gout is essentially caused by excessively high serum uric acid concentration, leading to the deposition of urate crystals in joints and tissues, causing inflammation and joint pain. To verify the therapeutic effects of B351, B352, and B353 carriers on gout, this example established an acute gout model in mice with hyperuricemia. The modeling method is referenced in the literature (Cairns LS et al. Uricase deficiency drives spontaneous metabolic syndrome and liver inflammation in mice [J]. Nature Medicine, 2014, 20 (11): 1283-1291.). 1.5 mg of sodium urate powder (MSU) was resuspended in 70 μL of sterile PBS to form a suspension, which was then injected subcutaneously into the foot of mice to simulate urate deposition.

[0178] The experimental mice were divided into 6 groups, with 8 mice in each group. The treatment methods for each group were as follows: Figure 12 As shown:

[0179] (1) WT+PBS group (blank control group): Wild-type mice were injected with saline via the tail vein at d-28 and sterile PBS was injected into the paw at d0. Acute gout modeling was not performed to exclude the effect of the injection operation itself on paw swelling.

[0180] (2) WT+MSU group (wild-type acute gout model group): wild-type mice were injected with saline via the tail vein at d-28 and with MSU suspension injected into the paw at d0.

[0181] (3) UOX-KO+MSU group (hyperuricemia acute gout model group): UOX-KO mice were injected with saline via the tail vein at d-28 and injected with MSU suspension at the same dose as the WT+MSU group at d0 to construct an acute gout inflammation model under the background of hyperuricemia.

[0182] (4) B351+MSU group (test drug administration group): UOX-KO mice were injected with B351 carrier (dose of 5E12 vg / kg) via the tail vein at d-28 and injected with MSU suspension of the same dose as the WT+MSU group via the paw to construct an acute gouty inflammation model for evaluating the pharmacological activity of B351 carrier.

[0183] (5) B352+MSU group (test drug administration group): UOX-KO mice were injected with B352 carrier (dose of 5E12 vg / kg) via the tail vein at d-28 and injected with MSU suspension of the same dose as the WT+MSU group via the paw to construct an acute gouty inflammation model for evaluating the pharmacological activity of B352 carrier.

[0184] (6) B353+MSU group (test drug administration group): UOX-KO mice were injected with B353 carrier (dose of 5E12 vg / kg) via the tail vein at d-28 and injected with MSU suspension of the same dose as the WT+MSU group via the paw to construct an acute gouty inflammation model for evaluating the pharmacological activity of B353 carrier.

[0185] On the day of modeling (d0, baseline before modeling) and at multiple time points d1, d2, d3, d4, d5, d6, d7, d8, d9, d10, d11, d12, d14, d16, and d18 after modeling, the thickness of the mouse's paw was measured using vernier calipers, and the degree of swelling was calculated. The formula was: Swelling degree on day n (dn) after modeling = (dn paw thickness - d0 paw thickness) / d0 paw thickness. The percentage of swelling (%) was used as the evaluation index.

[0186] The results are as follows Figure 13 As shown in the figure. Throughout the entire experimental period, the paw swelling of mice in the WT+PBS group remained at the baseline level, demonstrating that sterile PBS injection and the procedure itself did not induce paw swelling in mice. Mice in the WT+MSU group showed significant paw swelling on day 1 after modeling, with a peak value of 47.38±6.78%. The swelling then showed a continuous decreasing trend, decreasing to 1.48±0.98% by day 10, returning to the baseline level. Mice in the UOX-KO+MSU group showed significant paw swelling on day 1 after modeling, and the swelling continued to increase from day 1 to day 6, reaching a peak of 68.63±5.38% on day 6. The swelling decreased slowly from day 6 to day 18, and was not fully recovered by the experimental endpoint of day 18 (9.86±4.24%).

[0187] The B351+MSU, B352+MSU, and B353+MSU groups reached their peak values ​​on day 1, followed by a continuous decline; by day 11, all treatment groups had returned to baseline levels. These results indicate that the B351, B352, and B353 carriers can significantly accelerate the resolution of inflammatory swelling.

[0188] In summary, the B351, B352, and B353 carriers have therapeutic effects on acute gout induced by sodium urate crystals. B351, B352, and B353 can reduce paw swelling after an acute gout attack in hyperuricemic mice, accelerate the resolution of inflammatory swelling, and reverse the pathological phenotype of severe inflammation and prolonged course of acute gout in the context of hyperuricemia.

[0189] Those skilled in the art will understand that many and various modifications can be made without departing from the spirit of the invention. Therefore, it should be clearly understood that the form of the invention is merely illustrative and is not intended to limit the scope of the invention.

[0190] All publications, patent applications, patents, patent publications, database accession number sequences, and other references mentioned herein are incorporated in their entirety by reference for the teachings relating to the sentences and / or paragraphs presenting those references.

[0191] The foregoing is illustrative of the invention and should not be construed as limiting it. The invention is defined by the appended claims, including their equivalents.

Claims

1. A nucleic acid molecule encoding an amino acid sequence as shown in SEQ ID NO: 12, wherein the nucleotide sequence of the nucleic acid molecule has at least 98% identity with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, preferably at least 99% or 100% identity.

2. The nucleic acid molecule according to claim 1, wherein, The nucleic acid molecule contains the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2. Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. A transgenic expression cassette, comprising: Promoter, nucleic acid molecule as described in claim 1 or 2, PolyA; Preferably, the polyA is bGH polyA.

4. The transgenic expression cassette according to claim 3, wherein, The promoter is selected from: HCR-hAAT promoter, FA promoter shown in SEQ ID NO: 5, and FA1 promoter shown in SEQ ID NO:

6.

5. The transgenic expression cassette according to claim 3 or 4, wherein, The transgenic expression cassette also includes: a) Two ITRs located at both ends, preferably, each of the two ITRs is independently a normal ITR or a shortened ITR; preferably, the ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVDJ, avian AAV, bovine AAV, canine AAV, horse AAV, sheep AAV, goat AAV, or mouse AAV ITR; b) An intron located between the promoter and the nucleic acid molecule, preferably, the intron is SV40; c) The Kozak sequence located between the promoter and the nucleic acid molecule; and / or d) The WPRE sequence located between the nucleic acid molecule and the PolyA.

6. The transgenic expression cassette according to any one of claims 3 to 5, wherein, The nucleotide sequence of the transgenic expression cassette is shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:

11. Preferably, the nucleotide sequence of the transgenic expression cassette is shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:

11. More preferably, it is shown in SEQ ID NO:

11.

7. A gene delivery system comprising: The transgenic expression cassette and AAV capsid protein as described in any one of claims 3 to 6, Preferably, the AAV capsid protein is selected from: AAV1, AAV3, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ8, AAV-LK03, Anc80L65, AAVrh39, AAVrh43, AAVrh74, AAV2G9, AAV.PHP, AAV3B, and AAV843 capsid proteins; more preferably, the AAV capsid protein is AAV843 capsid protein.

8. The use of the nucleic acid molecule of claim 1 or 2, the transgenic expression cassette of any one of claims 3 to 6, or the gene delivery system of claim 7 in the preparation of a medicament for treating a disease. Preferably, the disease is selected from diseases that can be improved by lowering serum uric acid levels, including but not limited to hyperuricemia, gout, gouty arthritis, uric acid nephropathy, uric acid kidney stones, chronic kidney disease, tumor lysis syndrome, and metabolic diseases caused or aggravated by elevated serum uric acid.

9. A medicament comprising: a nucleic acid molecule as claimed in claim 1 or 2, a transgenic expression cassette as claimed in any one of claims 3 to 6 or a gene delivery system as claimed in claim 7, and optionally an excipient; Preferably, the drug is administered by systemic or local injection, such as by intravenous injection, portal vein injection, hepatic artery injection, intraperitoneal injection, subcutaneous injection or intramuscular injection, more preferably by intravenous injection; Preferably, the drug is an injectable preparation.

10. A method for delivering a nucleic acid molecule encoding a mammalian ancestral uricase into target cells, wherein the amino acid sequence of the mammalian ancestral uricase is shown in SEQ ID NO: 12, the method comprising: 1) The transgenic expression cassette of any one of claims 3 to 6 is packaged in AAV capsid protein to form the gene delivery system of claim 7; as well as 2) Bring the target cells into contact with the gene delivery system. Preferably, the target cell is an isolated cell.

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