A nucleic acid molecule for preparing a high-activity, long-acting uric acid oxidase, and a preparation method and application thereof

Abstract

The embodiments of the present specification provide a recombinant DNA molecule for preparing RNA, comprising elements operably linked and arranged in the following order in 5' to 3' direction: (a) an exon E2 fragment; (b) an internal ribosome entry site (IRES) fragment; (c) a 5' signal peptide element; (d) a gene fragment encoding a mutant urate oxidase; (e) a 3' end signal peptide element and (f) an exon E1 fragment; wherein the gene fragment encoding the urate oxidase comprises any one of SEQ ID NOs. 1-6, 10-12.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a nucleic acid molecule for preparing a highly active, long-acting uricase, its preparation method, and its application. Background Technology

[0002] Hyperuricemia is a disease state caused by purine metabolism disorders, leading to the accumulation of uric acid in the blood. Hyperuricemia is often closely related to gout, a chronic inflammatory disease caused by the deposition of sodium urate crystals. Uric acid oxidase (uricase) catalyzes the oxidation of uric acid to allantoin, playing a significant clinical role in lowering blood uric acid levels and treating gout and hyperuricemia.

[0003] Currently, exogenous uricase is used clinically as a biological agent to treat hyperuricemia and gout, but its main limitations lie in its short half-life and strong immunogenicity. Several methods, such as PEGylation and genetic engineering, have been developed to improve the stability and bioactivity of uricase. However, these methods still have certain limitations, such as a short half-life or unresolved immunogenicity issues.

[0004] Therefore, how to extend the half-life of uricase and reduce its immunogenicity through innovative methods remains a technical problem that urgently needs to be solved. Summary of the Invention

[0005] One embodiment of this specification provides a recombinant DNA molecule for preparing RNA. In some embodiments, the recombinant DNA molecule comprises elements operatively linked and arranged in the following order in the 5' to 3' orientation: (a) an exon E2 fragment; (b) an internal ribosome entry site (IRES) fragment; (c) a 5' signal peptide element; (d) a gene fragment encoding uricase; (e) a 3' signal peptide element; and (f) an exon E1 fragment; wherein the gene fragment encoding mutant uricase has at least 95% similarity to any one of SEQ ID NOs. 1-6, 10-12.

[0006] In some embodiments, the gene fragment encoding uricase includes at least one of a mutant uricase encoding sequence derived from Aspergillus flavus, a mutant uricase encoding sequence derived from pigs, and a mutant uricase encoding sequence derived from baboons.

[0007] In some embodiments, the amino acid mutation sites of the mutant uricase derived from Aspergillus flavus include at least one of K5R, K21R, K24S, K49S, K139Q, C104A, K115R, E137N, K139T, K190R, K204R, K218R, K290R, S297K, K299S, and K301R.

[0008] In some embodiments, the amino acid mutation sites of the porcine-derived mutant uricase include at least one of K9R, K27R, D34N, K36T, K41I, K54R, K55R, K82R, K85R, K103N, I106N, A108T, V125T, K126R, I146N, N148T, H200N, G202T, Q201N, R203T, K272N, E279T, K297R, and T301S, and the amino acid mutation sites of the baboon-derived mutant uricase include at least one of K9R, K54R, K55R, K82R, K85R, K126R, K272N, and K291R.

[0009] In some embodiments, the gene fragment encoding uricase is structurally optimized, and the optimized DNA molecule contains a gene fragment encoding uricase that includes any one of the sequences in SEQ ID NOs.1, 10-12.

[0010] In some embodiments, the nucleotide sequence of the E2 fragment has at least 95% similarity to SEQ ID NO.7, and the nucleotide sequence of the E1 fragment has at least 95% similarity to SEQ ID NO.8.

[0011] In some embodiments, the IRES fragment is derived from Taura syndrome virus, blood-sucking assassin bug virus, Leyle's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, grain constrictor aphid virus, reticuloendotheliosis virus, Forman poliovirus 1, soybean inchworm virus, Kashmir wasp virus, human rhinovirus 2, glass leafhopper virus-1, human immunodeficiency virus type 1, glass leafhopper virus-1, louse P virus, hepatitis C virus, hepatitis A virus, GB Hepatitis virus type 71, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, tea geometrid moth-like virus, encephalomyelitis virus (EMCV), fruit fly C virus, cruciferous tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, fruit fly antennae and legs Human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIFla, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, salivary virus, Coxsackievirus, bi-echovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian microRNA virus, turnip shrunkenness virus, aptamer of eIF4G, Coxsackievirus A (CVB1 / 2) or Coxsackievirus B3 (CVB3).

[0012] In some embodiments, the IRES fragment has at least 95% similarity to SEQ ID NO.9.

[0013] In some embodiments, the 5' signal peptide encoded by the 5' signal peptide element is used to guide the uricase into the endoplasmic reticulum to complete glycosylation modification, and the 3' signal peptide encoded by the 3' signal peptide element is used to transport the glycosylated uricase from the endoplasmic reticulum to the peroxisome.

[0014] In some embodiments, the amino acid sequence translated from the 5' signal peptide element includes any one of SEQ ID NOs.13, 24-28, and the amino acid sequence translated from the 3' signal peptide element includes any one of SEQ ID NOs.22-23.

[0015] In some embodiments, the recombinant DNA molecule further comprises a 5' homologous arm sequence and a 3' homologous arm sequence located between the E2 fragment and the E1 fragment, wherein the 5' homologous arm sequence has at least 95% similarity to SEQ ID NO.16 and the 3' homologous arm sequence has at least 95% similarity to SEQ ID NO.17.

[0016] In some embodiments, a start codon is connected before the 5' signal peptide element, and two stop codons are connected after the 3' signal peptide element.

[0017] In some embodiments, the stop codon is followed by a 3' untranslated region (3'UTR), the nucleotide sequence of which has at least 95% similarity to SEQ ID NO.18.

[0018] One embodiment of this specification provides an RNA molecule generated based on the recombinant RNA molecule as described above. In some embodiments, the RNA includes mRNA or circular RNA.

[0019] One embodiment of this specification provides a method for preparing an RNA molecule. In some embodiments, the method includes expressing the recombinant DNA molecule as described above in a host cell or a cell-free expression system.

[0020] One embodiment of this specification provides the use of RNA as described above in the preparation of a medicament for lowering uric acid in a subject or for treating diseases related to high uric acid levels. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of plasmid construction containing recombinant DNA molecules, as shown in some embodiments of this specification.

[0022] Figure 2 The results are based on the purification of RNA molecules shown in some examples of this specification;

[0023] Figure 3 This is a graph showing the enzyme activity assay results of a uricase mutant expressed in cells by a recombinant DNA molecule containing a gene fragment encoding a mutant uricase, as illustrated in some embodiments of this specification.

[0024] Figure 4 The results are the long-term effects and stability of uricase mutants expressed in cells by recombinant DNA molecules containing gene fragments encoding mutant uricase, as shown in some embodiments of this specification.

[0025] Figure 5 This is a graph showing the yield of uricase mutants according to some embodiments of this specification;

[0026] Figure 6 This is a diagram illustrating the effect of circular RNA expressing mutant uricase in mice, as shown in some embodiments of this specification. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0028] As shown in this disclosure and claims, unless the context clearly indicates otherwise, the words “a,” “an,” and / or “the” do not specifically refer to the singular form but may also include the plural form. The terms “comprising” and “including” imply only the inclusion of expressly identified steps and elements, which do not constitute an exclusive enumeration, and the method or apparatus may also include other steps or elements.

[0029] The flowcharts used in this invention illustrate the operations performed by the system in the embodiments of this invention. It should be understood that the preceding or following operation in the flowchart is not necessarily executed in exact order. Instead, the operations can be executed in reverse order or simultaneously. Furthermore, other operations can be added to the flowchart, or one or more operations can be removed from the flowchart.

[0030] definition

[0031] In this article, "intron" refers to a non-coding segment of a DNA sequence. "Exon" refers to a coding segment of a DNA sequence that can be transcribed and translated into a protein. A gene's DNA sequence can include both introns and exons. During transcription, the gene is transcribed into an intermediate molecule called premessenger RNA (or linear RNA). In premessenger RNA, introns are transcribed but not retained in the mature mRNA.

[0032] Although introns do not directly encode proteins, they may play an important role in gene expression regulation and evolution. Through regulation and splicing, cells produce a variety of proteins to adapt to different biological processes and environmental conditions.

[0033] In this article, "downstream exon" refers to the exon located after the intron in the premessenger RNA sequence corresponding to the gene's DNA sequence. Upstream exons are usually located before downstream exons. The terms "upstream" and "downstream" in this article refer to the spatial location of elements within a genome or RNA sequence. For example, "upstream" refers to the direction away from the intron, while "downstream" refers to the direction closer to the intron.

[0034] In this article, "transcription" refers to the process of synthesizing RNA using DNA molecules as templates. In cellular structures, DNA carries encoded genetic information. To effectively execute this genetic information within the cell, it needs to be copied from DNA into RNA molecules. This copying enables protein production or other functions during translation.

[0035] The circular RNAs (circRNAs) used in this paper are an important class of regulatory non-coding RNAs. Circular RNAs typically contain a closed circular structure and are generally unaffected by RNA exonucleases. Circular RNAs are generally stable and can regulate gene expression through a variety of mechanisms. Circular RNAs hold promise as therapeutic agents. In this disclosure, circular RNAs can express uricase in cells and can therefore be configured to lower uric acid levels.

[0036] The linear RNA used in this article is a single-stranded molecule composed of ribonucleic acid molecules with well-defined 5' and 3' ends. In some embodiments, linear RNA is typically synthesized based on a DNA template via transcription. In some embodiments, the synthesis of linear RNA involves post-transcriptional modifications such as splicing, capping, and tailing.

[0037] One embodiment of this specification provides a recombinant DNA molecule for preparing RNA. In some embodiments, the recombinant DNA molecule may include elements operatively linked and arranged in a 5' to 3' orientation in the following order:

[0038] (a) Exon E2 fragment;

[0039] (b) Internal ribosome entry site (IRES) fragment;

[0040] (c) 5' signal peptide element;

[0041] (d) Gene fragment encoding uricase;

[0042] (e) 3' signal peptide element; and

[0043] (f) Exon E1 fragment; wherein the gene fragment encoding uricase has at least 95% similarity to any one of SEQ ID NOs.1-6, 10-12.

[0044] In some embodiments, the DNA molecule sequence is a modified sequence. In some embodiments, modification may include at least one of RNA structure optimization, introduction and optimization of signal peptides, optimization of ubiquitination sites, and introduction and optimization of glycosylation sites.

[0045] In some embodiments, the gene segment encoding uricase is a mutated segment. The gene segment encoding mutated uricase refers to the coding region (or coding sequence) in the uricase gene that is translated to form the mutated uricase.

[0046] A mutation refers to a point mutation or sequence change in the amino acid sequence of uricase. For example, such mutations can include mutations targeting key amino acid residues (such as lysine, arginine, aspartic acid, etc.) in the catalytic reaction of uricase. Mutation types include, but are not limited to: point mutations: replacing one or more amino acid residues, such as mutating lysine (K) to arginine (R); insertion mutations: inserting additional amino acid residues into the gene sequence; and deletion mutations: deleting an amino acid residue at a specific position or a small segment of nucleotide sequence.

[0047] In some embodiments, mutations in uricase may include optimization of ubiquitination sites. In some embodiments, ubiquitination sites in uricase (e.g., K9, K41, K54, etc.) can be predicted using proteomics and optimized. Specifically, if these sites have adverse effects on the protein's structure or function (e.g., leading to excessive degradation or immunogenicity problems), they are replaced or masked through mutational design. By reducing the degradation potential of uricase, its stability in vivo and in vitro is further improved, and its half-life is prolonged, thereby enhancing the long-term efficacy of uricase in treating diseases such as hyperuricemia or gout.

[0048] In some embodiments, glycosylation sites may be further introduced into the gene sequence of uricase. These glycosylation sites are typically located at appropriate positions within the protein to enhance the stability and anti-degradation capacity of uricase through glycosylation modification, reduce its immunogenicity, and prolong its half-life in vivo. In this process, the selection and optimization of glycosylation sites can be tailored to the functional requirements of uricase to ensure that the optimized enzyme exhibits good biological performance.

[0049] In some embodiments, the uricase gene sequence can be optimized for thermodynamic / thermal stability (structural optimization). By altering specific amino acid sequences to improve protein stability, the thermal stability of uricase in both in vivo and in vitro environments can be enhanced. Through this optimization, uricase can maintain its structural integrity and enzymatic activity under high temperature, low pH, or other adverse conditions, thereby improving its operability and stability in clinical treatment.

[0050] The embodiments in this specification, through sequence optimization, such as the introduction of signal peptides, optimization of ubiquitination sites, introduction of glycosylation sites, and enhancement of thermal stability, can improve the structure, function, stability, and long-term efficacy of uricase, thereby enhancing its therapeutic effect and reducing potential side effects and immune responses, thus giving it greater application potential in clinical applications.

[0051] In some embodiments, the urate oxidase gene may be derived from animals, plants, microorganisms, etc. In some embodiments, the urate oxidase gene may be derived from Aspergillus flavus, pigs (Susscrofa), mice, rats, bovines, baboons (Papio hamadryas), dogs, rabbits, cynomolgus monkeys (Macaca fascicularis), night monkeys (Aotus trivirgatus), rhesus monkeys (Rheus Macaque), zebrafish (Danio rerio), Arthrobacter globiformis, and Dictyostelium discoideum, etc.

[0052] In some embodiments, the gene fragment encoding uricase may be derived from a chimeric sequence, such as a pig-baboon chimeric (PBC) sequence, a pig-horse chimeric sequence, etc. Here, a chimeric sequence refers to a sequence composed of two or more uricase gene sequences.

[0053] In some embodiments, the gene fragment encoding uricase includes at least one of an aspergillus-derived uricase-encoding sequence, a porcine-derived uricase-encoding sequence, and a baboon-derived uricase-encoding sequence.

[0054] In some embodiments, the wild-type uricase sequence from Aspergillus flavus is shown in SEQ ID NO.14, the wild-type uricase sequence from pigs is shown in SEQ ID NO.20, and the wild-type uricase sequence from baboons is shown in SEQ ID NO.21.

[0055] In some embodiments, the gene fragment encoding uricase is obtained through structural optimization.

[0056] In some embodiments, structural optimization may include thermodynamic stability optimization. Structural optimization of the gene fragment encoding uricase can be achieved in various ways. In some embodiments, structural optimization of the gene fragment encoding uricase can be achieved through the following steps: analyzing the thermodynamic stability of the target uricase based on protein three-dimensional structure prediction and molecular dynamics simulation, and identifying unstable regions in its structure (e.g., flexible loop regions or easily unfoldable fragments); experimentally verifying the thermostability of the optimized uricase (e.g., determining its melting temperature (Tm value) by differential scanning calorimetry (DSC) or thermal denaturation experiments); evaluating its enzyme activity and function; and determining the optimized RNA and DNA sequences based on the amino acid sequence of the target uricase with high enzyme activity.

[0057] In some embodiments, the gene fragment encoding uricase has at least 95%, 96%, 97%, 98%, or 99% similarity to any one of SEQ ID NOs.1,10-12.

[0058] In some embodiments, the gene fragment encoding uricase has at least 95%, 96%, 97%, 98%, or 99% similarity to any one of SEQ ID NOs.11-12.

[0059] In some embodiments, the gene fragment encoding uricase is a structurally optimized porcine uricase-encoding sequence.

[0060] In some embodiments, the coding sequence for urate oxidase in pigs is SEQ ID NO.11.

[0061] In some embodiments, the gene fragment encoding uricase is a structurally optimized uricase-encoding sequence derived from baboons.

[0062] In some embodiments, the coding sequence of uricase derived from baboons is SEQ ID NO.12.

[0063] In some embodiments, the gene fragment encoding uricase includes at least one of a mutant uricase encoding sequence derived from Aspergillus flavus, a mutant uricase encoding sequence derived from pigs, and a mutant uricase encoding sequence derived from baboons.

[0064] In some embodiments, the gene fragment encoding uricase has at least 95%, 96%, 97%, 98%, or 99% similarity to any one of SEQ ID NOs.1-6, 10.

[0065] In some embodiments, the gene fragment encoding uricase is a mutant uricase encoding sequence derived from Aspergillus flavus.

[0066] In some embodiments, the amino acid mutation sites of the mutant uricase derived from Aspergillus flavus include at least one of K5R, K21R, K24S, K49S, K139Q, C104A, K115R, E137N, K139T, K190R, K204R, K218R, K290R, S297K, K299S, and K301R.

[0067] In some embodiments, the amino acid mutation sites of the mutant uricase from Aspergillus flavus include C103A, S297K, K299S and K301R, and the nucleotide sequence of the gene fragment encoding the uricase is SEQ ID NO.1 or SEQ ID NO.10.

[0068] In some embodiments, the amino acid mutation sites of the mutant uricase from Aspergillus flavus are K5R, K21R, K24R, C104A, K115R, K190R, K290R, S297K, K299S and K301R, and the nucleotide sequence of the gene fragment encoding uricase is SEQ ID NO.2.

[0069] In some embodiments, the amino acid mutation sites of the mutant uricase from Aspergillus flavus are K5R, K21R, C104A, K115R, E137N, K139T, K190R, K290R, S297K, K299S, and K301R, and the nucleotide sequence of the gene fragment encoding the uricase is SEQ ID NO.3.

[0070] In some embodiments, the gene fragment encoding uricase is a pig-derived mutant uricase coding sequence.

[0071] In some embodiments, the amino acid mutation sites of the porcine-derived mutant uricase include at least one of K9R, K27R, D34N, K36T, K41I, K54R, K55R, K82R, K85R, K103N, I106N, A108T, V125T, K126R, I146N, N148T, H200N, G202T, Q201N, R203T, K272N, E279T, K297R, and T301S.

[0072] In some embodiments, the amino acid mutation sites of the porcine-derived mutant uricase are K9R, K54R, K55R, K82R, K85R, K103N, K126R, K272N, and T301S, and the nucleotide sequence of the gene fragment encoding the uricase is SEQ ID NO.4.

[0073] In some embodiments, the amino acid mutation sites of the porcine-derived mutant uricase are K9R, K54R, K55R, K82R, K85R, K103N, K126R, Q201N, R203T, K272N, and T301S, and the nucleotide sequence of the gene fragment encoding the uricase is SEQ ID NO.5.

[0074] In some embodiments, the gene fragment encoding uricase is a mutant uricase encoding sequence derived from baboons.

[0075] In some embodiments, the amino acid mutation sites of the baboon-derived mutant uricase include at least one of K9R, K54R, K55R, K82R, K85R, K126R, K272N, and K291R.

[0076] In some embodiments, the amino acid mutation sites of the baboon-derived mutant uricase are K9R, K54R, K55R, K82R, K85R, K126R, K272N, and K291R, and the nucleotide sequence of the gene fragment encoding the uricase is SEQ ID NO.6.

[0077] In some embodiments, the E2 fragment and the E1 fragment originate from the same genome.

[0078] In some embodiments, the E2 fragment refers to the exon sequence located downstream of the intron. That is, the downstream exon portion retained after splicing of the intron region.

[0079] In some embodiments, the E1 fragment refers to an exon sequence located upstream of an intron, typically an exon portion near the transcription start site. The E1 fragment is one of the sequences retained after transcription and is eventually translated into a specific protein within the cell.

[0080] This document does not limit the genomic origin of the E2 and E1 fragments, and they can be selected according to the needs of specific embodiments. Exemplarily, the E1 and E2 fragments can each originate from mouse genes. In some embodiments, the intron fragment, E1, and E2 fragments can be cloned into an expression vector and transfected into mouse cells. In the transfected cells, the introns are correctly removed, and the exons E1 and E2 are accurately spliced, ultimately expressing the mutant uricase. In some embodiments, the E1 and E2 fragments originate from the *Anabaena* sp. gene. In some embodiments, the E1 fragment is an upstream exon fragment located in the type I intron of *Anabaena* sp., and the E2 fragment is a downstream exon fragment located in the type I intron of *Anabaena* sp. In some embodiments, RNA molecules capable of expressing the mutant uricase can be prepared based on template DNA containing the E1 fragment, the E2 fragment, and optionally, the intron fragment, through in vitro transcription. The RNA molecule can be linear or circular. The E1 and E2 fragments are accurately spliced ​​during in vitro transcription and circularization. In some embodiments, the nucleotide sequence of the E2 fragment has at least 95% similarity to SEQ ID NO. 7, and the nucleotide sequence of the E1 fragment has at least 95% similarity to SEQ ID NO. 8. In some embodiments, the nucleotide sequence of the E2 fragment has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO. 7, and the nucleotide sequence of the E1 fragment has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO. 8.

[0081] In some embodiments, the nucleotide sequence of the E2 fragment is SEQ ID NO.7, and the nucleotide sequence of the E1 fragment is SEQ ID NO.8.

[0082] The IRES fragment used in this article is a DNA sequence that encodes a translatable RNA fragment. This fragment then mediates translation within the transcribed RNA molecule via an internal ribosome entry site, allowing the ribosome to initiate translation independently of the traditional 5' cap structure. The IRES sequence itself does not contain a translation promoter; instead, it interacts with translation factors through its specific secondary structure, directly guiding the ribosome to bind to a specific site on the RNA, thereby initiating protein synthesis.

[0083] In some embodiments, the IRES fragment is derived from Taura syndrome virus, blood-sucking assassin bug virus, Leyle's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, grain constrictor aphid virus, reticuloendotheliosis virus, Forman poliovirus 1, soybean inchworm virus, Kashmir wasp virus, human rhinovirus 2, glass leafhopper virus-1, human immunodeficiency virus type 1, glass leafhopper virus-1, louse P virus, hepatitis C virus, hepatitis A virus, GB Hepatitis virus type 71, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, tea geometrid moth-like virus, encephalomyelitis virus (EMCV), fruit fly C virus, cruciferous tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, fruit fly antennae and legs Human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIFla, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, salivary virus, Coxsackievirus, bi-echovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian microRNA virus, turnip shrunkenness virus, aptamer of eIF4G, Coxsackievirus A (CVB1 / 2) or Coxsackievirus B3 (CVB3).

[0084] In some embodiments, the IRES fragment is derived from CVB3.

[0085] In some embodiments, the IRES fragment has at least 95% similarity to SEQ ID NO. 9. In some embodiments, the IRES fragment has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO. 9. In some embodiments, the IRES fragment is SEQ ID NO. 9.

[0086] In some embodiments, the IRES fragment includes ribosome recognition sequences pIRES1-pIRES10.

[0087] In some other embodiments, the IRES fragment may contain IRES sequences from other viral sources (such as EMCV, adenovirus, etc.) or optimized IRES sequences to adapt to different expression systems and needs.

[0088] In some embodiments, the recombinant DNA molecule further includes a signal peptide element that encodes a signal peptide for prompting the secretion of uricase outside the cell, wherein the signal peptide element is located between the IRES fragment and the gene fragment encoding the mutant uricase.

[0089] In some embodiments, the signal peptides include interleukin-2 (IL-2) signal peptide, human leukocyte antigen (HLA) signal peptide, leucine-rich α-2 glycoprotein 1 (LRG1) signal peptide, cholinergic receptor nicotinic α1 subunit (CHRNA1) signal peptide, apolipoprotein B (APOB) signal peptide, cystatin D (CST5) signal peptide, galactosylceramidinase (GALC) signal peptide, gel sol (GSN) signal peptide, glycoprotein Ib platelet subunit α (GP1BA) signal peptide, granzyme B (GZMB) signal peptide, and SE. RPING1 signal peptide, interleukin-12 subunit α (IL-12A) signal peptide, interleukin-2 (IL-10) signal peptide, interleukin-1 receptor-like 1 (IL1RL1) signal peptide, insulin receptor (INSR) signal peptide, cytotoxic cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1) signal peptide, kallikrein-associated peptidase 14 (KLK14) signal peptide, prolactin (LACRT) signal peptide, lymphocyte activation gene-3 (LAG3) signal peptide, or any combination thereof.

[0090] In some embodiments, the signal peptide may include an N-terminal (amino-terminal) signal peptide and a C-terminal (carboxyl-terminal) signal peptide. In some embodiments, the N-terminal signal peptide may include at least one of HB-EGF, an endoplasmic reticulum (ER) signal peptide, and a PTS2 signal peptide. In some embodiments, the C-terminal signal peptide may include a PTS1 signal peptide.

[0091] In some embodiments, a signal peptide element can be linked to the 5' end of the gene segment encoding uricase. Alternatively, a signal peptide element can be linked to the 3' end of the gene segment encoding uricase. In some embodiments, the 5' signal peptide element encodes a 5' signal peptide that guides the uricase into the endoplasmic reticulum to complete glycosylation modification, and the 3' signal peptide element encodes a 3' signal peptide that transports the glycosylated uricase from the endoplasmic reticulum to the peroxisome.

[0092] In some embodiments, the amino acid sequence translated from the 5' signal peptide element has at least 95% similarity to any one of SEQ ID NOs. 13, 24-28. In some embodiments, the amino acid sequence translated from the 5' signal peptide element has at least 95%, 96%, 97%, 98%, or 99% similarity to any one of SEQ ID NOs. 13, 24-28. In some embodiments, the amino acid sequence translated from the 5' signal peptide element is any one of SEQ ID NOs. 13, 24-28.

[0093] In some embodiments, the amino acid sequence translated from the 3' signal peptide element has at least 95% similarity to any one of the sequences in SEQ ID NOs. 22-23. In some embodiments, the amino acid sequence translated from the 3' signal peptide element has at least 95%, 96%, 97%, 98%, or 99% similarity to any one of the sequences in SEQ ID NOs. 22-23. In some embodiments, the amino acid sequence translated from the 3' signal peptide element is any one of the sequences in SEQ ID NOs. 22-23.

[0094] In some embodiments, when uricase is introduced into the glycosylation site, if the glycosylation site in the tertiary structure may affect the binding of the PTS1 signal peptide to the peroxisome membrane receptor PEX5, then the PTS2 signal peptide can be added to the N-terminus of uricase to adjust the PTS2 sequence and increase hydrophobicity, so as to ensure that uricase can be successfully glycosylated through the ER pathway.

[0095] In some embodiments, an ER signal peptide or a PTS2 signal peptide may be linked to the left side of the uricase derived from Aspergillus flavus, and a PTS1 signal peptide may be linked to the right side of the uricase derived from Aspergillus flavus. In some embodiments, the gene fragment encoding uricase with the added signal peptide element has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO. 15. In some embodiments, the gene fragment encoding uricase with the added signal peptide element is SEQ ID NO. 15. In some embodiments, an ER signal peptide or a PTS2 signal peptide may be linked to the left side of the porcine uricase oxidase, and a PTS1 signal peptide may be linked to the right side of the porcine uricase oxidase. In some embodiments, the gene fragment encoding uricase with the added signal peptide element has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO. 19. In some embodiments, the gene fragment encoding uricase with the added signal peptide element is SEQ ID NO. 19.

[0096] This specification describes an embodiment in which a specific signal peptide (such as HB-EGF or PTS2) is added to the N-terminus of uricase. This signal peptide guides the immature uricase peptide chain into the endoplasmic reticulum (ER) for glycosylation modification. PTS2 not only enables ER localization but also facilitates the targeted transport of uricase to peroxidase. In this design, the processing site of the signal peptide is also optimized to ensure that the N-terminal sequence can be cleaved during uricase maturation without affecting its functional integrity.

[0097] This specification describes an embodiment in which a specific signal peptide, PTS1, is added to the C-terminus of the uricase gene. This signal peptide enables the targeted transport of uricase into the peroxisome via binding to the peroxisome receptor (PEX5) and transport through the PEX14 membrane pores. In this design, the processing site of the signal peptide is also optimized to ensure that the N-terminal sequence can be cleaved during uricase maturation without affecting its functional integrity.

[0098] In some embodiments, the recombinant DNA molecule may further include a 5' homologous arm sequence and a 3' homologous arm sequence located between the E2 fragment and the E1 fragment.

[0099] The 5' homologous arm sequence is typically located at the 5' end of a DNA molecule; the 3' homologous arm sequence is typically located at the 3' end of a DNA molecule. Both the 5' and 3' homologous arm sequences are designed and inserted into recombinant DNA molecules to perform homologous recombination.

[0100] In some embodiments, the 5' homologous arm sequence has at least 95% similarity to SEQ ID NO. 16, and the 3' homologous arm sequence has at least 95% similarity to SEQ ID NO. 17. In some embodiments, the 5' homologous arm sequence has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO. 16, and the 3' homologous arm sequence has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO. 17. In some embodiments, the 5' homologous arm sequence is SEQ ID NO. 16, and the 3' homologous arm sequence is SEQ ID NO. 17.

[0101] In some embodiments, a start codon is attached before the 5' signal peptide element.

[0102] In some embodiments, at least one stop codon is attached after the 3' signal peptide element.

[0103] In some embodiments, the 3' signal peptide element is followed by two stop codons.

[0104] In some embodiments, the stop codon is followed by a 3' untranslated region (3'UTR), the nucleotide sequence of which has at least 95% similarity to SEQ ID NO. 18. In some embodiments, the nucleotide sequence of the 3'UTR has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO. 18. In some embodiments, the nucleotide sequence of the 3'UTR is SEQ ID NO. 18.

[0105] In some embodiments, recombinant DNA molecules can be generated by: constructing a recombinant plasmid containing a DNA molecule sequence; digesting the recombinant plasmid with a type II restriction endonuclease or a type II blunt-end restriction endonuclease to obtain nucleic acid molecules.

[0106] Type IIS restriction enzymes may include Acu I, Alw I, Bae I, Bbs I, BbV I, Bcc I, BceA I, BcgI, BciV I, Bmr I, Bpm I, BpuE I, BsaX I, BseR I, Bsg I, BsmAI, BsmBI-v2, BsmF1, Bsm I, BspCN I, BspM I, BspQ I, BsrD I, Bsr I, BtgZ I, BtsC I, Btsl-v2, Btslmut I, CspC I, EarI, Eci I, Esp3 I, Fau I, Fok I, Hga I, Hph I, HpyA V, Mbo II, Mly I, Mme I, Mnl I, NmeAIII, PaqC I, Ple I, Sap I and SfaN At least one of I.

[0107] Type II blunt-ended restriction endonucleases may include at least one of Afe I, Alu I, BsaA I, BstU I, BstZ17 I, DraI, EcoRV, Fsp I, Hae III, Hpa I, Hinc II, MSc I, MspA1 I, Nae I, Nru I, Pme I, Pm II, Pvu II, Rsa I, Sca I, Sfo I, Sma I, SnaB I, Ssp I, Stu I, or Swa I.

[0108] One embodiment of this specification provides a method for optimizing the uricase gene sequence, aiming to obtain a gene fragment encoding a mutant uricase by modifying specific sites of the uricase gene, thereby improving its stability and biological activity and reducing its immunogenicity. In some embodiments, the gene fragment encoding the mutant uricase can be obtained through the following steps.

[0109] Amino acid sequence alignment and conservation analysis: First, multiple sequence alignment was performed on the amino acid sequence of uricase before mutation to determine its highly conserved positions and active sites. By comparing the amino acid sequences of uricases from different species or sources, highly conserved amino acid residues (e.g., residues near the active site) were identified during evolution.

[0110] A highly conserved site refers to the location of an amino acid sequence that remains highly consistent across different species. An active site refers to the amino acid residue or region within the uricase molecule that directly participates in the catalytic reaction. For example, in uricases from humans and other mammals, the amino acid residues located at positions 40 to 50 can serve as conserved catalytic active sites.

[0111] Avoid cleavage sites of hydrolases: Hydrolases (e.g., lysosomes) typically cleave proteins at certain specific sequences, leading to loss of enzyme function or instability. Therefore, when optimizing the amino acid fragment of uricase, it is necessary to avoid cleavage sites that may be recognized by hydrolases.

[0112] For example, sequences containing x-phe-x-arg-gln (XFXRQ) and gln-arg-x-phe-x (QRXFX) (x = any amino acid) are susceptible to lysosomal cleavage ubiquitination site prediction and evaluation: further prediction of possible ubiquitination sites in the amino acid sequence of uricase.

[0113] Ubiquitination sites are amino acid residues on uricase molecules that can covalently bind to ubiquitin molecules. Ubiquitination is an important regulatory mechanism in protein degradation, modulating the stability and function of uricase. Predicting ubiquitination sites requires assessing whether these sites affect the structure or conservation of the active site. In some embodiments, ubiquitination sites in uricase may include K9, K41, K54, etc.

[0114] Based on the ubiquitination site assessment results, the amino acid sequence is modified: If the predicted ubiquitination site is located at the active site or has a significant impact on conservation, the original amino acid sequence of uricase is preserved to avoid potential negative effects from mutations; if the predicted ubiquitination site does not significantly affect the structure or conservation of the enzyme's active site, the thermodynamics, thermal stability, structural changes, and hydrogen bond changes of uricase after mutation can be predicted, and glycosylation masking can be performed based on the amino acid sequence (e.g., by introducing glycosylation modifications to mask the influence of ubiquitination sites) to reduce the impact of ubiquitination on enzyme function. In some embodiments, glycosylation modifications include N-linked glycosylation, O-linked glycosylation, sialylation, fucosylation, and GalNAc modification.

[0115] Modification of N-linked glycosylation sites:

[0116] Introducing NXT-type glycosylation sites (where N represents any amino acid, T represents threonine, and X represents any amino acid except P) at specific amino acid positions in uricase can increase glycosylation modification at these positions. Glycosylation can increase protein stability and may reduce the adverse effects of ubiquitination by altering protein conformation or surface properties.

[0117] For example, introducing an NXT-type glycosylation site at position 137 of uricase may mask the ubiquitination reaction at that site with glycosylation, thereby reducing the degradation effect of ubiquitination on the enzyme.

[0118] O-linked glycosylation modification (O-glycosylation):

[0119] O-linked glycosylation typically occurs at serine or threonine residues of amino acids. This modification adds sugar molecules to the surface of proteins, enhancing their stability and preventing ubiquitination and degradation. O-linked glycosylation can also affect protein structure, making it more adaptable to the environment within the organism.

[0120] In some embodiments, an O-linked glycosylation site can be introduced near position 24 of uricase, and a glycosyl group (such as glucosamine or galactose) can be added to the amino acid residue at this site, thereby improving the stability and function of the protein.

[0121] Sialylation modification:

[0122] Sialylation is a common form of glycosylation, particularly the addition of sialic acid molecules (such as sialic acid or N-acetylneuraminic acid) to the end of N- or O-glycosylation. This modification introduces a negative charge into the protein, enhancing its stability and reducing its recognition by the immune system, thereby prolonging its half-life in vivo.

[0123] In some embodiments, adding sialic acid molecules to the N-linked glycosylation modification end of uricase can enhance the negative charge of the protein, reduce its likelihood of being recognized by the immune system, and thus improve its stability in vivo.

[0124] Fucosylation modification:

[0125] Fucose-rich glycosylation modifications can affect protein stability, resistance to degradation, and cell receptor binding capacity. Fucose is a sugar molecule that frequently appears on cell surfaces and during the glycosylation of some secretory proteins. Introducing fucose-rich modifications can help reduce the degradation by uricase and increase its activity in vivo.

[0126] In some embodiments, introducing fucose glycosylation modifications (e.g., increasing the fucose-rich glycans) at certain N-linked or O-linked glycosylation sites of uricase can improve its stability in vivo and reduce potential immunogenic responses.

[0127] GalNAc glycosylation modification (N-acetylgalactosamine glycosylation):

[0128] GalNAc glycosylation modification can be used to alter the stability and structure of uricase.

[0129] In some embodiments, introducing N-acetylgalactosamine glycosylation modification near the O-linked glycosylation site of uricase can increase the glycosylation density at that site, thereby enhancing its stability and reducing the negative effects of ubiquitination.

[0130] Design and optimization of candidate nucleotide sequences: After optimizing the amino acid sequence, candidate amino acid sequences for mutant uricase and corresponding candidate nucleotide sequences (e.g., candidate RNA sequences) are obtained by introducing appropriate glycosylation sites. In some embodiments, the RNA sequence can be further optimized. For example, the candidate RNA sequence can be further optimized using MFE values ​​and CAI to ensure efficient intracellular expression.

[0131] The MFE value refers to the free energy of RNA secondary structure, which reflects the most stable conformation of the RNA molecule.

[0132] The CAI value, or Coding Adaptability Index, measures the degree of optimization of a target gene to improve its translation efficiency in host cells.

[0133] In some embodiments, the MFE value of the optimized RNA sequence can be calculated to ensure that the RNA molecule folds into the most stable structure in the cell, thus avoiding low expression efficiency due to instability of RNA secondary structure.

[0134] In some embodiments, the optimized RNA sequence obtained through the above steps can be used to synthesize a gene fragment encoding a mutant uricase. This gene fragment can be cloned into a suitable expression vector for further protein expression and production, or for therapeutic applications.

[0135] This specification provides examples of a highly stable, highly active, and low-immunogenic mutant uricase and its preparation method. The mutant uricase can be used to treat hyperuricemia, gout, and other diseases, showing promising clinical application prospects.

[0136] In some embodiments, the recombinant DNA molecule may be a vector, and as used herein, a vector is a tool used to carry exogenous DNA fragments and perform transcription reactions within cells to produce RNA.

[0137] Vectors are typically circular DNA molecules, such as plasmids or viruses (e.g., adenovirus, adeno-associated virus), bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc. These vectors have the ability to self-replicate, can replicate independently within cells, and can also carry foreign genes, such as protein-coding genes, RNA genes, etc.

[0138] In some embodiments, the vector may be designed to include specific start codons, regulatory elements, and stop codons to transcribe exogenous DNA in the cell into RNA, which may be protein-coding mRNA or other non-coding RNA.

[0139] In some embodiments, an in vitro transcription template can be obtained based on the above-described vector, and RNA can be formed in an in vitro transcription reaction based on the in vitro transcription template.

[0140] The in vitro transcription template can be obtained by various methods. For example, it can be obtained directly by artificial in vitro synthesis. In some embodiments, the in vitro transcription template can be obtained by constructing a plasmid and performing PCR amplification, or by cutting the plasmid with a restriction endonuclease.

[0141] One embodiment of this specification provides an RNA molecule, which is generated based on the aforementioned recombinant DNA molecule.

[0142] In some embodiments, the RNA includes linear RNA or circular RNA.

[0143] In some embodiments, mutant uricase can be produced using a circular RNA expression system. Circular RNAs exhibit long-term stability and high expression efficiency. By using a circular RNA expression system, the expression efficiency and stability of uricase can be significantly improved.

[0144] One embodiment of this specification provides a method for preparing RNA molecules, the method comprising: expressing any of the recombinant DNA molecules described above in a host cell or a cell-free expression system.

[0145] In some embodiments, the expression includes: performing an in vitro transcription reaction on the recombinant DNA molecule to obtain linear RNA.

[0146] In some embodiments, the expression includes: performing an in vitro transcription reaction on the recombinant DNA molecule to obtain linear RNA; and self-circulating the linear RNA to generate circular RNA.

[0147] In some embodiments, the method further includes purifying the RNA molecule.

[0148] In some embodiments, recombinant DNA molecules are produced through in vitro synthesis.

[0149] In some embodiments, the recombinant DNA molecule can be generated by: constructing a recombinant plasmid including a promoter and a nucleic acid molecule sequence; using the recombinant plasmid as a template, and employing forward and reverse primers at the ends of the E1 fragment to obtain the nucleic acid molecule through PCR amplification.

[0150] In some embodiments, the method further includes encapsulating the circular RNA with lipid nanoparticles (LNPs). LNP encapsulation can enhance the advantages of circular RNA in protein production. LNPs are state-of-the-art nanoparticle carriers that can be used to target specific cells with endogenous or exogenous ligands by encapsulating circular RNA. The endocytosis of LNPs destabilizes the endosome membrane and releases the circular RNA into the cytoplasm. LNPs can address many of the challenges associated with circular RNA molecules, making them less susceptible to degradation and promoting cellular uptake.

[0151] In some embodiments, encapsulating circular RNA with LNP further includes: dissolving LNP in ethanol to obtain an LNP solution; dissolving circular RNA in sodium acetate solution to obtain a circular RNA solution; and passing the LNP solution and the circular RNA solution through a microfluidic device to obtain LNP-encapsulated circular RNA.

[0152] The LNP may include SM102, DME-PEG2000, DSPC, and cholesterol. In some embodiments, the molar ratio of SM102:DSPC:cholesterol:DME-PEG2000 is 30-60:3-20:25-50:0.2-5, or 35-58:5-18:28-45:0.5-3, or 40-55:8-15:30-43:0.8-2, etc. In some embodiments, the molar ratio of SM102:DSPC:cholesterol:DME-PEG2000 is 50:10:38.5:1.5.

[0153] In some embodiments, the molar N / P ratio of the LNP solution to the circular RNA solution is in the range of 2-8:1, or 2-7:1, or 2-6:1, or 2-5:1, etc. In some embodiments, the molar N / P ratio of the LNP solution to the circular RNA solution is 3:1, or 2:1, or 4:1, or 5:1, or 6:1, or 7:1, or 8:1. As used herein, the molar N / P ratio (also referred to as N / P ratio, N:P ratio, or NP) is defined as the ratio of amino groups in the ionizable lipids of the LNP solution to phosphate groups on the circular RNA backbone.

[0154] It is worth noting that, in addition to LNPs, other circular RNA-based drug delivery systems can be used, such as gold nanoparticles (AuNPs), engineered exosomes, but are not limited to these.

[0155] This specification provides an embodiment of a method for treating a subject with a disease associated with high uric acid levels, the method comprising administering a pharmaceutically acceptable amount of RNA to the subject.

[0156] As used in this article, “subject” refers to either a human or an animal. Typically, an animal is a vertebrate, such as primates (e.g., chimpanzees, cynomolgus monkeys, spider monkeys, and macaques), rodents (e.g., mice, rats, marmots, ferrets, rabbits, and hamsters), or livestock or prey (e.g., cattle, horses, pigs, deer, bison, buffalo, and felines). In some embodiments, a subject is a mammal, such as a primate, like a human.

[0157] As used herein, the term "pharmaceutically acceptable amount" refers to the amount of circular RNA that provides therapeutic benefit in treating diseases with high uric acid levels or in lowering uric acid levels, for example, an amount that provides, for example, a statistically significant reduction in serum uric acid. The determination of a pharmaceutically acceptable amount is entirely within the competence of those skilled in the art. Generally, a pharmaceutically acceptable amount can vary depending on the subject's medical history, age, condition, sex, and the severity and type of the subject's medical condition, as well as the administration of other pharmaceutically active agents.

[0158] In some embodiments, the circular RNA may be administered to the subject at doses ranging from 0.1 μg / kg to 200 μg / kg, 0.1 μg / kg to 150 μg / kg, 0.1 μg / kg to 100 μg / kg, 1 μg / kg to 150 μg / kg, etc. In some embodiments, the circular RNA may be administered to the subject every two days, every four days, weekly, every two weeks, every two weeks, every three days, every four days, every two weeks, every two days, every four days, every two weeks, every two days, every four days, every two weeks, every four days, every two weeks, every four days... or at any time interval within 3 years.

[0159] As used herein, the term "hyperuricemia-related disease" refers to a disease, disorder, or medical condition that can cause or is directly or indirectly caused by high uric acid levels. Exemplary diseases include hyperuricemia, uric acid stones, gout, cardiovascular disease, chronic kidney disease, atherosclerosis, and any combination thereof. In some embodiments, the disease includes hyperuricemia.

[0160] One embodiment of this specification provides the use of RNA in the preparation of a medicament for lowering uric acid in a subject or for treating diseases related to high uric acid levels.

[0161] The present invention is illustrated by the following embodiments, but is not intended to limit the invention. Example Example 1: Sequence optimization encoding uricase

[0162] Amino acid sequences of wild-type uricase oxidase from Aspergillus flavus were mutated, including the following mutations: C103A, S297K, K299S and K301R (mutated sequences corresponding to SEQ ID NO.1 or SEQ ID NO.10), K5R; K21R; K24R; C104A; K115R; K190R; K290R; S297K; K299S; K301R (mutated sequences corresponding to SEQ ID NO.2) and K5R; K21R; K24R; C104A; K115R; E137N; K139T; K190R; K290R; S297K; K299S; K301R (mutated sequences corresponding to SEQ ID NO.3).

[0163] The wild-type porcine (Pig / Sus scrofa) uricase was structurally optimized to obtain the nucleotide sequence shown in SEQ ID NO. 11. Amino acid mutations were then performed on the wild-type porcine (Pig / Sus scrofa) uricase, including the following mutations: K9R; K54R; K55R; K82R; K85R; K103N; K126R; K272N; T301S (mutated sequences corresponding to SEQ ID NO. 4), K9R; K54R; K55R; K82R; K85R; K103N; K126R; Q201N; R203T; K272N; T301S (mutated sequences corresponding to SEQ ID NO. 5).

[0164] The wild-type uricase from baboon (PAPHA) was structurally optimized to obtain the nucleotide sequence shown in SEQ ID NO.12. Amino acid mutations were then performed on the wild-type uricase from baboon (PAPHA) to include the following mutations: K9R; K54R; K55R; K82R; K85R; K126R; K272N; K291R (mutated sequences correspond to SEQ ID NO.6). Example 2: Preparation of RNA expressing mutant uricase

[0165] 1. Preparation of DNA templates capable of expressing mutant uricase genes

[0166] This embodiment uses a gene containing a type I intron of the genus *Anabaena* sp. as an example to design E2 and E1 fragments, but it is not limited to *Anabaena* sp. genes; it can also be E2 and E1 fragments from other genes of the same origin. Between the 5' homologous arm sequence A (shown in SEQ ID NO. 16) and the 3' homologous arm sequence B (shown in SEQ ID NO. 17) of the vector, the insert fragment and the fragment from the vector are ligated together using sequence recombination. After transformation into competent cells and pressure selection, a plasmid expressing the uricase gene is obtained.

[0167] A schematic diagram of the construction of a plasmid expressing the uricase gene is shown below. Figure 1 As shown, the sequence includes: E2 (SEQ ID NO. 7); IRES sequence (SEQ ID NO. 9); 5' signal peptide element (translated amino acid sequence as shown in any of SEQ ID NOs. 13, 24-28); uric acid oxidase coding region (Uricase CDS, any of SEQ ID NOs. 1-6, 10-12); 3' signal peptide element (translated amino acid sequence as shown in any of SEQ ID NOs. 22-23); terminator (2*TGA); 3'UTR (SEQ ID NO. 18); and E1 (SEQ ID NO. 8).

[0168] The specific process is as follows:

[0169] (1) Amplification using F / R primers;

[0170] Reaction system: F primer (10mM), 2.5μL; R primer (10mM), 2.5μL; plasmid, 10ng; 2×Taq Mix, 25μL; H2O to 50μL.

[0171] Reaction conditions: 95℃, 5 min; 95℃, 10 s; 55℃, 10 s; 35 cycles; 72℃, 2 min; 72℃, 5 min; 4℃, ∞.

[0172] (2) DNA gel electrophoresis was used to recover the target PCR product;

[0173] Prepare a 2% DNA agarose gel, electrophoresis at 120V for 30 min, and recover the PCR product using the Molpure DNA Purification Kit to obtain the DNA template.

[0174] 2. In vitro transcription

[0175] This example uses the nearshore T7 High Yield RNA Transcription kit (Cat.#E131) as an example. Add the following components in sequence: 2 μL of 10×Transcription Buffer; 1.5 μL each of a 100 mM mixture of ATP / GTP / CTP / UTP; 500 ng - 1 μg of DNA template; 1 μL of T7 Enzyme Mix; and nuclease-free water to a final volume of 20 μL. Gently mix each component using a pipette (or vortex), and briefly centrifuge to collect the reaction solution at the bottom of the tube. Incubate at 37°C for 1 hour, then at 45°C for 1 hour.

[0176] 3. DNase I digestion

[0177] The template DNA was removed by treating with DNase I for 15 minutes.

[0178] Reaction conditions per tube: Add 2-4 U DNase I to the in vitro transcription reaction product, mix with a pipette tip, and react at 37°C for 15 min to digest the transcribed DNA template and obtain linear RNA molecules.

[0179] In some embodiments, the DNA template may further include intron fragments, and the intron fragments, E1 fragments, and E2 fragments may originate from the same genome. For example, the intron fragments, E1 fragments, and E2 fragments may originate from the genus *Anabaena*. When the template DNA contains intron fragments, after digestion with DNase I in the in vitro transcription reaction, 20 μL of 10× cyclization buffer and H2O are added to a final volume of 200 μL, and the mixture is incubated at 50-55°C for 20 min to generate circular RNA.

[0180] 4. RNA purification

[0181] (1) Purification of Oligo dT

[0182] Wash with 0.1M NaOH aqueous solution, then rinse with ultrapure water. Equilibrate with 50mM Tris, 250mM NaCl, pH 8.0. Then load and equilibrate the sample using a 1mL pre-packed Proteomix POR-dT20 column and collect the flow-through fraction, which is the purified RNA fraction. Finally, rinse the column with ultrapure water to elute the bound fraction.

[0183] (2) SEC purification

[0184] The column was equilibrated with 10 mM PB, 150 mM NaCl, and pH 6.0. The sample to be purified was then loaded onto a SRTSEC-1000A column (30 × 300 mm, 5 μm), and the target peak was collected. The purification results are shown below. Figure 2 As shown. Example 3: Cell transfection and determination of uricase activity from lysate products

[0185] The reagents involved in this embodiment include: Hieff mRNA Transfection Reagent(40809ES03); Serum-depleted transfection medium (Opti-MEM) (Yuanpei, L530KJ); RIPA Lysis Buffer (Yuanye, R21237); Western and IP cell lysis buffer (Beyotime, P0013); PBS buffer (Seville, G4202); PMSF (Seville, G2008-1ML); 2X SDS-PAGE protein loading buffer (Beyotime, P0015B).

[0186] The specific steps are as follows:

[0187] (1) Cell transfection

[0188] Cells were seeded in 6-well plates, with an appropriate number of cells seeded in each well (the exact number of cells depends on the cell type and experimental requirements). The cells were cultured in complete culture medium (such as DMEM or RPMI 1640 containing 10% fetal bovine serum). The cells were then incubated at 37°C in a 5% CO2 incubator until the cell confluence reached approximately 80%.

[0189] Preparation before transfection: Prepare the transfection complex: In a sterile 1.5 mL centrifuge tube, add Hieff The mRNA Transfection Reagent (40809ES03) was mixed with an appropriate amount of Opti-MEM medium (source culture, L530KJ), gently mixed, and incubated at room temperature for 5 minutes. In another sterile 1.5mL centrifuge tube, the purified RNA prepared in Example 2 was mixed with Opti-MEM medium and gently mixed.

[0190] Slowly add the diluted RNA solution to the diluted transfection reagent, mix gently, and let stand at room temperature for 15-20 minutes to form a transfection complex.

[0191] Transfection procedure:

[0192] Before transfection, replace the cell culture medium in the 6-well plate with 2 mL of Opti-MEM medium (serum-reduced medium). Add the prepared transfection complex dropwise to each well of the 6-well plate, gently shaking the plate to ensure even distribution. Incubate the cells at 37°C and 5% CO2 for 4-6 hours, then replace with complete medium and continue culturing for 48 hours.

[0193] (2) Cell lysis

[0194] Gently disperse the cell pellet, then add 150 μL of pre-chilled lysis buffer and PMSF (final concentration 1 mM) to a 6-well plate for lysis. Place the plate on ice, gently disperse the cell suspension, and lyse for 15 min in a 4°C freezer or ice-water mixture.

[0195] (3) Centrifugation

[0196] Centrifugation conditions: 12000 rpm, 4℃, 10 min. Separate the supernatant.

[0197] (4) Protein concentration determination

[0198] Protein concentration was determined using the BCA kit (Adamas Life, E8053-100T-PKG).

[0199] (5) Preparation of uric acid working solution

[0200] Uric acid was dissolved in sodium borate buffer (1M, pH 8.5) to prepare a 0.6mM uric acid working solution.

[0201] (6) Detection of urate oxidase activity

[0202] At 25℃ and pH 8.5, the amount of enzyme that converts 1 μmol of uric acid to allantoin per minute is defined as one unit of activity (U). 200 μL of 0.6 mM uric acid solution was placed in a test tube, and 20 μL of protein (original concentration, 1 / 5 concentration, and 1 / 10 concentration, experimental group numbers H / M / L respectively) was added. Uric acid (UA) concentration was measured using a phosphotungstic acid colorimetric method (LEAGENE, TC1197) at 5 min, 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, and 360 min. The control group was measured at the above time points, and also at 0 min. The concentration was calculated using the formula U = ΔUA / T (ΔUA is the change in uric acid concentration, and T is the reaction time). The final result was compared between groups using enzyme activity (U / mg). Results are as follows: Figure 3 As shown, the porcine mutants exhibited 9.1% and 12.5% ​​higher activity than the wild-type, respectively. pWU is the original sequence of porcine uricase, pMU1 is the porcine uricase mutant corresponding to the mutant sequence SEQ ID NO.4, and pMU2 is the porcine uricase mutant corresponding to the mutant sequence SEQ ID NO.5.

[0203] After determining the RNA optimized sequence using the same method, the relative activity of the aflatoxin-derived mutant was 1.82-fold and 2.95-fold higher than that of the original sequence. Figure 4 (Corresponding to optimized sequence 1, corresponding to optimized sequence 2), the enzyme activity of the mutant after introducing the NxT site decreased slightly by 20±5% (not shown in the figure).

[0204] Using the same method, the optimized sequence derived from baboons showed a 1.68-fold increase in activity compared to the original sequence, while the deubiquitinated mutant showed a 1.36-fold increase in activity. (Not shown in the figure) Example 4: Determination of the long-term effectiveness and stability of urate oxidase mutants

[0205] The RNA-optimized uricase was placed in a 37°C incubator for 0.5 h, 1 h, 2 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h. Samples were then removed, and the uricase activity was measured according to the method described in Example 3. The activity and enzyme activity retention rate were compared. Results are as follows: Figure 4 As shown. By Figure 4 It can be seen that optimized sequence 1 (corresponding to SEQ ID NO.1) and optimized sequence 2 (corresponding to SEQ ID NO.10) still have significant activity after 144 hours. Example 5: Detection of the yield of uricase mutant

[0206] Take an appropriate amount of the lysed protein sample (usually 20-30 μg, adjust the amount according to experimental needs), add an equal volume of 5X SDS-PAGE protein loading buffer (Beyotime, P0015B), and mix gently. Ensure that the protein and loading buffer are thoroughly mixed to avoid abnormal banding during electrophoresis.

[0207] Heat the mixed sample in a boiling water bath for 10 minutes to fully denature the proteins. Ensure the sample tube caps are tightly closed during heating to prevent sample evaporation or contamination. After heating, immediately place the sample on ice to cool for 2-3 minutes to avoid protein degradation.

[0208] Briefly centrifuge (approximately 10,000 rpm, 30 seconds) to collect droplets on the tube wall, ensuring the sample is completely concentrated at the bottom of the tube.

[0209] Electrophoresis detection: Prepare an SDS-PAGE gel (select an appropriate gel concentration based on the molecular weight of the target protein, such as a 10% or 12% separating gel). Add the prepared protein sample to the gel wells, along with a pre-stained protein molecular weight marker (such as a PageRuler Prestained Protein Ladder) as a reference. Add 1X SDS-PAGE electrophoresis buffer to the electrophoresis tank and perform electrophoresis detection.

[0210] like Figure 5 As shown, the protein yield of porcine uricase mutants was increased. Changes in protein expression levels can be quantitatively analyzed by comparing electrophoretic band intensities or Western blot signal intensities.

[0211] The yield of stable mutants derived from aflatoxin was also increased when tested in the same manner (not shown in the figure).

[0212] Based on the experimental results, the enzyme activity of the porcine mutant was significantly increased compared to the wild type (increased by 9.1% and 12.5%, respectively). Combined with the increased protein yield, it is speculated that the increased activity may be related to the optimization of protein expression efficiency, i.e., the enzyme activity per unit protein (specific enzyme activity) increases synchronously with the increase in expression level. However, after RNA structure optimization, the protein expression efficiency of aflatoxin-derived uricase was significantly increased, and the activity decayed less over time. The mutant, on the other hand, exhibited a contradictory trend: although protein yield was significantly increased, its enzyme activity was lower than the original mutant (decreased by 20±5%). This suggests that the introduction of glycosylation sites may weaken the enzyme's catalytic efficiency through conformational changes or interference with active sites—although the increase in protein yield can increase the total enzyme amount to some extent, the loss of enzyme activity per unit cannot be fully compensated, ultimately leading to a decrease in overall activity. It is worth noting that the deubiquitination, glycosylation, and expression enhancement properties of Aspergillus flavus mutants may endow them with a longer protein retention time in vivo. This advantage may compensate for the lack of enzyme activity in vitro, thus demonstrating potential value in applications such as drug long-acting properties. Example 6 In vivo experiment

[0213] Experimental subjects: Uox-Ko mice, male, 4-5 weeks old (purchased from Biocytogen), acclimatized in the laboratory for 7 days, and then grouped, numbered and injected.

[0214] Injection method: Circular RNA (aspergillus flavus source, containing C103A, S297K, K299S and K301R mutations, optimized RNA structure, corresponding to SEQ ID NO.1) was encapsulated in a lipid nanoparticle (LNP) delivery system and injected via the tail vein of mice.

[0215] Blood collection method: One orbital blood sample is taken before injection, another orbital blood sample is taken 3 days later, and then another eye blood sample is taken every 7 days.

[0216] Single test result: Take the average of 2-3 tests as the test result.

[0217] The results are as follows Figure 6 As shown, the circular RNA expressing uricase exhibits very strong efficacy in mice for the first 7 days, followed by a very long maintenance period (21 days).

[0218] As described above, the basic concepts have been fully explained, and it will be quite clear to those skilled in the art after reading this detailed disclosure that the above detailed disclosure is intended to be illustrative only and is not intended to be limiting. Although not explicitly stated herein, various changes, improvements, and modifications can be made by those skilled in the art. These changes, improvements, and modifications are intended to be proposed in this disclosure and fall within the spirit and scope of the exemplary embodiments of this disclosure.

[0219] Furthermore, certain terms have been used to describe embodiments of this disclosure. For example, the terms "an embodiment," "an embodiment," and / or "some embodiments" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. Therefore, it is important to emphasize and understand that references to "an embodiment" or "an embodiment" or "alternative embodiment" two or more times in various parts of this disclosure do not necessarily all refer to the same embodiment. Moreover, specific features, structures, or characteristics may be suitably combined in one or more embodiments of this disclosure.

[0220] Furthermore, the order of the processing elements or sequences described, or the use of numbers, letters or other names, is not intended to limit the claimed process and method to any order other than that which may be specified in the claims.

[0221] In some embodiments, the numbers used to describe quantities, characteristics, etc., of certain embodiments of this application should be understood to be modified in certain circumstances by the terms “about,” “approximately,” or “substantially.” For example, “about,” “approximately,” or “substantially” may represent a variation of ±20% of the value described, unless otherwise stated. Thus, in some embodiments, the numerical parameters listed in the written description and appended claims are approximate values ​​that may vary depending on the desired characteristics to be obtained in a particular embodiment. In some embodiments, numerical parameters should be interpreted based on the number of significant figures reported and the application of common rounding techniques. Although the numerical ranges and parameters illustrating a broad range of some embodiments of this application are approximate values, the numerical values ​​listed in the specific examples are reported as precisely as possible.

[0222] Every patent, patent application, patent application publication, and other material (e.g., articles, books, specifications, publications, documents, things, and / or the like) cited herein is incorporated in its entirety by reference for all purposes, except for any related litigation history, any content inconsistent with or conflicting with this document, or any content that may limit the widest scope of the claims relating to this document now or thereafter. For example, if there is any inconsistency or conflict between the use of descriptions, definitions, and / or terms relating to any incorporated material and the use of descriptions, definitions, and / or terms relating to this document, the use of descriptions, definitions, and / or terms in this document shall prevail.

[0223] Finally, it should be understood that the embodiments disclosed herein are merely illustrative of the principles of the embodiments of this application. Other modifications may be employed within the scope of this application. Therefore, as examples, but not limitations, alternative configurations of the embodiments of this application may be utilized in accordance with the teachings herein. Consequently, the embodiments of this application are not limited to the embodiments shown and described.

Claims

1. A recombinant DNA molecule for preparing RNA, characterized in that, The recombinant DNA molecule comprises elements operatively linked and arranged in the following order at a 5' to 3' orientation: (a) Exon E2 fragment; (b) Internal ribosome entry site (IRES) fragment; (c) 5' signal peptide element; (d) Gene fragment encoding uricase; (e) 3' signal peptide element; and (f) Exon E1 fragment; wherein the gene fragment encoding uricase is selected from any one of SEQ ID NOs.1,4-6,10,12.

2. The recombinant DNA molecule according to claim 1, characterized in that, The gene fragment encoding uricase has been structurally optimized, and the gene fragment encoding uricase in the optimized DNA molecule is selected from any one of SEQ ID NOs. 1, 10, and 12.

3. The recombinant DNA molecule according to claim 1, characterized in that, The nucleotide sequence of the E2 fragment is SEQ ID NO. 7, and the nucleotide sequence of the E1 fragment is SEQ ID NO.

8.

4. The recombinant DNA molecule according to claim 1, wherein the IRES fragment is derived from Taura syndrome virus, blood-sucking assassin bug virus, Leyle's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, cereal constrictor aphid virus, reticuloendotheliosis virus, Forman poliovirus 1, soybean looper virus, Kashmir bee virus, human rhinovirus 2, human immunodeficiency virus type 1, glass leafhopper virus-1, louse P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, tea looper-like virus, encephalocarditis virus (EMCV), fruit fly C virus, cruciferous tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, fruit Fly antennae and legs, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIFla, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, salivary viruses, Coxsackieviruses, bi-echoviruses, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian microRNA virus, turnip shrunkenness virus, aptamers of eIF4G, Coxsackievirus A (CVB1 / 2) or Coxsackievirus B3 (CVB3).

5. The recombinant DNA molecule according to claim 1, wherein the IRES fragment is SEQ ID NO.

9.

6. The recombinant DNA molecule according to claim 1, characterized in that, The 5' signal peptide element encodes a 5' signal peptide that guides the uricase into the endoplasmic reticulum to complete glycosylation modification. The 3' signal peptide element encodes a 3' signal peptide that transports the glycosylated uricase from the endoplasmic reticulum to the peroxisome. The amino acid sequence translated from the 5' signal peptide element is selected from any one of SEQ ID NOs. 13, 24-28, and the amino acid sequence translated from the 3' signal peptide element is selected from any one of SEQ ID NOs. 22-23.

7. The recombinant DNA molecule according to claim 1, characterized in that, The recombinant DNA molecule further comprises a 5' homologous arm sequence and a 3' homologous arm sequence located between the E2 fragment and the E1 fragment, wherein the 5' homologous arm sequence is SEQ ID NO. 16 and the 3' homologous arm sequence is SEQ ID NO.

17.

8. The recombinant DNA molecule according to claim 5, characterized in that, The 5' signal peptide element is preceded by a start codon, and the 3' signal peptide element is followed by two stop codons.

9. The recombinant DNA molecule according to claim 8, characterized in that, The stop codon is followed by a 3' untranslated region (3'UTR), the nucleotide sequence of which is SEQ ID NO.

18.

10. An RNA molecule, characterized in that, The RNA is generated based on the recombinant DNA molecule of any one of claims 1-9, and the RNA is linear RNA or circular RNA.

11. A method for preparing an RNA molecule, characterized in that, The method includes expressing any one of the recombinant DNA molecules according to claims 1-9 in a host cell or a cell-free expression system.

12. The use of RNA according to claim 10 in the preparation of a medicament for treating diseases related to high uric acid levels, wherein the diseases related to high uric acid levels are hyperuricemia, uric acid stones, and gout.

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