Nucleic acid molecule for preparing high-activity and long-acting urate oxidase as well as preparation method and application of nucleic acid molecule

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nucleic acid molecule for preparing a highly active, long-acting uricase, and a preparation method and application thereof. Background Art

[0002] Hyperuricemia is a disease state in which uric acid accumulates in the blood due to purine metabolism disorders. Hyperuricemia is often closely related to the occurrence of gout, a chronic inflammatory disease caused by the deposition of sodium urate crystals. Urate oxidase (uricase) can catalyze the oxidation of uric acid to produce allantoin, and has important clinical significance in reducing blood uric acid concentration and treating gout and hyperuricemia.

[0003] Currently, exogenous uricase is used clinically as a biological agent for the treatment of hyperuricemia and gout, but its main limitations are its short half-life and strong immunogenicity. There are some methods, such as PEG modification and genetic engineering, aimed at improving the stability and biological activity of uricase. However, these methods still have certain limitations, such as short half-life or failure to effectively solve the immunogenicity problem.

[0004] Therefore, how to prolong the half-life of urate oxidase and reduce immunogenicity through innovative methods remains a technical problem that needs to be solved urgently. Summary of the invention

[0005] One of the embodiments of the present specification provides a recombinant DNA molecule for preparing RNA. In some embodiments, the recombinant DNA molecule comprises elements that are operably linked and arranged in the following order from 5' to 3': (a) an exon E2 fragment; (b) an internal ribosome entry site (IRES) fragment; (c) a 5'-terminal signal peptide element; (d) a gene fragment encoding uricase; (e) a 3'-terminal signal peptide element and (f) an exon E1 fragment; wherein the gene fragment encoding a 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 comprises at least one of a mutant uricase coding sequence derived from Aspergillus flavus, a mutant uricase coding sequence derived from pig, and a mutant uricase coding sequence derived from baboon.

[0007] In some embodiments, the amino acid mutation site of the mutant uricase derived from Aspergillus flavus includes 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 site of the mutant uricase from pig includes 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 site of the mutant uricase from baboon includes 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 gene fragment encoding uricase in the optimized DNA molecule includes any one of 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, Reye's encephalomyelitis virus, simian virus 40, fire ant virus 1, cereal aphid virus, reticuloendotheliosis virus, Forman polio virus 1, soybean looper virus, Kashmir bee virus, human rhinovirus 2, green leafhopper virus-1, human immunodeficiency virus type 1, green leafhopper virus-1, louse P virus, hepatitis C virus, hepatitis A virus, GB Hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid-like virus, encephalomyocarditis virus (EMCV), fruit fly C virus, crucifer 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 ringspot virus, swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, fruit fly antennapedia , human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1a, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, salivirus, coxsackievirus, double 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 picornavirus, turnip shrivelled disease virus, an aptamer for 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 the 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' homology arm sequence and a 3' homology arm sequence located between the E2 fragment and the E1 fragment, wherein the 5' homology arm sequence has at least 95% similarity to SEQ ID NO.16, and the 3' homology arm sequence has at least 95% similarity to SEQ ID NO.17.

[0016] In some embodiments, the 5' signal peptide element is preceded by a start codon and the 3' signal peptide element is followed by two stop codons.

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

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

[0019] One of the embodiments 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 of the embodiments of the present specification provides a use of the RNA as described above in the preparation of a drug for reducing uric acid in a subject or treating a disease associated with high uric acid levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of constructing a plasmid containing a recombinant DNA molecule according to some embodiments of this specification;

[0022] Figure 2 It is the purification result of RNA molecules shown in some embodiments of this specification;

[0023] Figure 3 It is a diagram showing the result of enzyme activity assay of a uricase mutant expressed in a cell by a recombinant DNA molecule comprising a gene fragment encoding a uricase mutant as shown in some embodiments of the present specification;

[0024] Figure 4 It is the result of the long-term effect and stability test of the uricase mutant expressed in cells by the recombinant DNA molecule containing the gene fragment encoding the mutant uricase as shown in some embodiments of this specification;

[0025] Figure 5 This is a graph for detecting the production of urate oxidase mutants according to some embodiments of this specification;

[0026] Figure 6 This is a diagram showing the effect of circular RNA expressing mutant urate oxidase in mice according to some embodiments of this specification. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] As shown in the present disclosure and claims, unless the context clearly indicates an exception, the words "a", "an" and / or "the" do not specifically refer to the singular form, but may also include the plural form. The terms "include" and "comprise" and the like only imply the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0029] The flowchart used in the present invention can illustrate the operations performed by the system in the embodiment of the present invention. It should be understood that the previous operation or the next operation in the flowchart is not necessarily performed accurately in order. On the contrary, each operation can be performed in reverse order or simultaneously. In addition, other operations can be added to the flowchart, or one or more operations can be removed from the flowchart.

[0030] definition

[0031] As used herein, "intron" refers to a non-coding segment in a DNA sequence. "Exon" refers to a coding segment in a DNA sequence that can be transcribed and translated into a part of a protein. The DNA sequence of a gene can include introns and exons. During transcription, a gene is transcribed into an intermediate molecule, which is called pre-messenger RNA (or linear RNA). In the pre-messenger RNA, introns are transcribed, but are not retained in 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 diverse proteins to adapt to different biological processes and environmental conditions.

[0033] As used herein, "downstream exon" refers to the exon located after the intron in the pre-messenger RNA sequence corresponding to the gene DNA sequence. The upstream exon is usually the exon located before the downstream exon. "Upstream" and "downstream" used herein refer to the spatial position of an element in a genome or RNA sequence. For example, "upstream" refers to the direction away from the intron, while "downstream" refers to the direction close to the intron.

[0034] As used herein, "transcription" refers to the process of synthesizing RNA using a DNA molecule as a template. In the cell structure, DNA carries the encoded biological genetic information. In order to effectively execute the biological genetic information in the cell, the biological genetic information in the DNA needs to be copied into the RNA molecule. This replication enables the production of proteins or other functions in the translation process.

[0035] Circular RNA (circular RNA, or circRNA) used herein is an important class of regulatory non-coding RNA. Circular RNA generally comprises a closed circular structure and is generally not affected by RNA exonucleases. Circular RNA is generally stable in nature and can regulate gene expression through a variety of mechanisms. Circular RNA is expected to become a therapeutic agent. In the present disclosure, circular RNA is able to express urate oxidase in cells and can therefore be configured to reduce uric acid levels.

[0036] Linear RNA used herein is a single-stranded molecule consisting of a ribonucleic acid molecule having a clear 5' end and 3' end structure. In some embodiments, linear RNA is generally synthesized based on a DNA template by a transcription process. In some embodiments, the synthesis process of linear RNA undergoes post-transcriptional modifications such as splicing, capping and tailing.

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

[0038] (a) Exon E2 fragment;

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

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

[0041] (d) a gene fragment encoding urate oxidase;

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

[0043] (f) Exon E1 fragment; wherein the gene fragment encoding urate oxidase 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, the 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 fragment encoding uricase is a mutated fragment. The gene fragment encoding a mutant uricase refers to the coding region (or coding sequence) in the uricase gene that is translated to form the mutant uricase.

[0046] Mutation refers to a point mutation or sequence change in the amino acid sequence of uricase. For example, the mutation may include mutations of 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 mutation: replacement of one or more amino acid residues, such as mutating lysine (K) to arginine (R), etc.; insertion mutation: insertion of additional amino acid residues in the gene sequence; and deletion mutation: deletion of amino acid residues or a short nucleotide sequence at a specific position.

[0047] In some embodiments, the mutation of the uricase can include optimizing ubiquitination sites. In some embodiments, ubiquitination sites (e.g., K9, K41, K54, etc.) in uricase can be predicted by proteomics, and these sites are optimized. Specifically, if these sites play an adverse effect (such as causing excessive degradation or immunogenicity problems) in the structure or function of the protein, these sites are replaced or shielded by mutation design. By reducing the degradation potential of uricase, its stability in vivo and in vitro is further improved, and its half-life is extended, thereby enhancing the long-term efficacy of uricase in treating diseases such as hyperuricemia or gout.

[0048] In some embodiments, glycosylation sites can be further introduced into the gene sequence of uricase. These glycosylation sites are usually located at appropriate positions of the protein to enhance the stability and anti-degradation ability 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 adjusted according to the functional requirements of uricase to ensure that the optimized enzyme has good biological properties.

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

[0050] The embodiments of this specification can improve the structure, function, stability and long-term effectiveness of uricase through sequence optimization, such as introduction of signal peptide, optimization of ubiquitination site, introduction of glycosylation site and enhancement of thermal stability, thereby improving the therapeutic effect of uricase and reducing possible side effects and immune responses, thereby making it have higher application potential in clinical applications.

[0051] In some embodiments, the uricase gene may be derived from animals, plants, microorganisms, etc. In some embodiments, the uricase gene may be derived from Aspergillus flavus, Susscrofa, Mouse, Rat, Bovine, Papio hamadryas, Dog, Rabbit, Macaca fascicularis, Aotus trivirgatus, Rhesus macaque, Danio rerio, Arthrobacter globiformis, 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 (also known as pig-baboon chimera, PBC) sequence, a pig-horse chimeric sequence, etc. The chimeric sequence here refers to a sequence composed of two or more uricase gene sequences.

[0053] In some embodiments, the gene fragment encoding uricase comprises at least one of a uricase coding sequence derived from Aspergillus flavus, a uricase coding sequence derived from pig, and a uricase coding sequence derived from baboon.

[0054] In some embodiments, the wild-type sequence of uricase derived from Aspergillus flavus is shown as SEQ ID NO.14, the wild-type sequence of uricase derived from pig is shown as SEQ ID NO.20, and the wild-type sequence of uricase derived from baboon is shown as 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 gene fragments encoding uricase can be achieved in a variety of ways. In some embodiments, structural optimization of gene fragments encoding uricase can be achieved by the following steps: based on protein three-dimensional structure prediction and molecular dynamics simulation, the thermodynamic stability of the target uricase is analyzed, and unstable regions in its structure are identified, such as flexible loop regions or easily unfolded fragments), the thermal stability of the optimized uricase is experimentally verified (such as by differential scanning calorimetry (DSC) or thermal denaturation experiments to determine its melting temperature (Tm value)), its enzyme activity and function are evaluated, and the amino acid sequence of the target uricase with high enzyme activity is determined to optimize the RNA and DNA sequence.

[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 uricase coding sequence of porcine origin.

[0060] In some embodiments, the coding sequence of porcine uricase is SEQ ID NO.11.

[0061] In some embodiments, the gene fragment encoding uricase is a structurally optimized uricase coding sequence from baboon.

[0062] In some embodiments, the baboon-derived uricase coding sequence is SEQ ID NO.12.

[0063] In some embodiments, the gene fragment encoding uricase comprises at least one of a mutant uricase coding sequence derived from Aspergillus flavus, a mutant uricase coding sequence derived from pig, and a mutant uricase coding sequence derived from baboon.

[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 coding sequence derived from Aspergillus flavus.

[0066] In some embodiments, the amino acid mutation site of the mutant uricase derived from Aspergillus flavus includes 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 derived from Aspergillus flavus include C103A, S297K, K299S and K301R, and the nucleotide sequence of the gene fragment encoding uricase is SEQ ID NO.1 or SEQ ID NO.10.

[0068] In some embodiments, the amino acid mutation sites of the mutant uricase derived 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 derived from Aspergillus flavus are K5R, K21R, C104A, K115R, E137N, K139T, K190R, K290R, S297K, K299S, K301R, and the nucleotide sequence of the gene fragment encoding uricase is SEQ ID NO.3.

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

[0071] In some embodiments, the amino acid mutation sites of the mutant uricase from pig origin 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 pig-derived mutant uricase are K9R, K54R, K55R, K82R, K85R, K103N, K126R, K272N and T301S, and the nucleotide sequence of the gene fragment encoding uricase is SEQ ID NO.4.

[0073] In some embodiments, the amino acid mutation sites of the pig-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 uricase is SEQ ID NO.5.

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

[0075] In some embodiments, the amino acid mutation site of the baboon-derived mutant uricase includes 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 uricase is SEQ ID NO.6.

[0077] In some embodiments, the E2 segment and the E1 segment are 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 the intron region is spliced.

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

[0080] The genomic sources of the E2 fragment and the E1 fragment are not limited herein and can be selected according to the needs of a specific embodiment. Exemplarily, the E1 and E2 fragments can be respectively from mouse genes. In certain embodiments, the intron fragments, E1 and E2 fragments can be cloned into expression vectors and transfected in mouse cells. In the transfected cells, the introns are correctly removed, the exon E1 and E2 fragments are accurately spliced, and the mutant uricase is finally expressed. In certain embodiments, the E1 and E2 fragments are from the Anabaena sp. gene. In certain embodiments, the E1 fragment is an upstream exon fragment located in the I type intron of the Anabaena sp., and the E2 fragment is an downstream exon fragment located in the I type intron of the Anabaena sp. In certain embodiments, an RNA molecule capable of expressing the mutant uricase can be prepared based on a template DNA by in vitro transcription reaction, and the template DNA comprises an E1 fragment, an E2 fragment, and an optional intron fragment. The RNA molecule can be linear or circular. The E1 fragment and the E2 fragment are accurately spliced ​​during in vitro transcription and cyclization. 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 can encode an RNA fragment that can be translated, and then in the transcribed RNA molecule, through the internal ribosome entry site, mediates the ribosome to start translation without relying on the traditional 5' cap structure. The IRES sequence itself does not contain a translation promoter, but interacts with translation factors through its specific secondary structure, directly guiding the ribosome to bind to a specific position of the RNA, thereby starting protein synthesis.

[0083] In some embodiments, the IRES fragment is derived from Taura syndrome virus, blood-sucking assassin bug virus, Reye's encephalomyelitis virus, simian virus 40, fire ant virus 1, cereal aphid virus, reticuloendotheliosis virus, Forman polio virus 1, soybean looper virus, Kashmir bee virus, human rhinovirus 2, green leafhopper virus-1, human immunodeficiency virus type 1, green leafhopper virus-1, louse P virus, hepatitis C virus, hepatitis A virus, GB Hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid-like virus, encephalomyocarditis virus (EMCV), fruit fly C virus, crucifer 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 ringspot virus, swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, fruit fly antennapedia , human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1a, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, salivirus, coxsackievirus, double 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 picornavirus, turnip shrivelled disease virus, an aptamer for 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 the ribosome recognition sequences pIRES1-pIRES10.

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

[0088] In some embodiments, the recombinant DNA molecule further comprises a signal peptide element, wherein the signal peptide element encodes a signal peptide for promoting the secretion of uricase to the outside of 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 peptide includes an interleukin-2 (IL-2) signal peptide, a human leukocyte antigen (HLA) signal peptide, a leucine-rich alpha-2 glycoprotein 1 (LRG1) signal peptide, a cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) signal peptide, apolipoprotein B (APOB) signal peptide, a cystatin D (CST5) signal peptide, a galactosylceramidase (GALC) signal peptide, a gelsolin (GSN) signal peptide, a glycoprotein Ib platelet subunit alpha (GP1BA) signal peptide, a granzyme B (GZMB) signal peptide, a SE signal peptide. RPING1 signal peptide, interleukin-12 subunit alpha (IL-12A) signal peptide, interleukin-2 (IL-10) signal peptide, interleukin 1 receptor-like 1 (IL1RL1) signal peptide, insulin receptor (INSR) signal peptide, killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1) signal peptide, kallikrein-related peptidase 14 (KLK14) signal peptide, prolactin (LACRT) signal peptide, lymphocyte activation gene-3 (LAG3) signal peptide, etc., 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 may be connected to the 5' end of the gene fragment encoding uricase. A signal peptide element may also be connected to the 3' end of the gene fragment encoding uricase. In some embodiments, the 5' end signal peptide encoded by the 5' end signal peptide element is used to guide the uricase to enter the endoplasmic reticulum to complete the glycosylation modification, and the 3' end signal peptide encoded by the 3' end signal peptide element is used to transport 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 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 SEQ ID NOs. 22-23. In some embodiments, the amino acid sequence translated from the 3' signal peptide element is any one of SEQ ID NOs. 22-23.

[0094] In some embodiments, when introducing uricase oxidase into a glycosylation site, if the glycosylation site in the tertiary structure may affect the binding of the PTS1 signal peptide to the peroxisomal membrane receptor PEX5, in this case, a PTS2 signal peptide can be added to the N-terminus of the uricase oxidase, and the PTS2 sequence can be adjusted to increase the hydrophobicity to ensure that the uricase oxidase can be successfully glycosylated through the ER pathway.

[0095] In some embodiments, an ER signal peptide or a PTS2 signal peptide can be connected to the left of the uricase derived from Aspergillus flavus, and a PTS1 signal peptide can be connected to the right of the uricase derived from Aspergillus flavus. In some embodiments, the gene fragment encoding uricase oxidase 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 oxidase with the added signal peptide element is SEQ ID NO.15. In some embodiments, an ER signal peptide or a PTS2 signal peptide can be connected to the left of the porcine uricase, and a PTS1 signal peptide can be connected to the right of the porcine uricase. 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] In the embodiment of this specification, a specific signal peptide (such as HB-EGF or PTS2) is added to the N-terminus of uricase, and the signal peptide can guide the immature uricase peptide chain into the endoplasmic reticulum ER to achieve glycosylation modification. Among them, PTS2 can not only achieve ER localization, but also enable uricase oxidase to be transported to the peroxisome in a directional manner. In this design, the processing site of the signal peptide is also optimized to ensure that the N-terminal sequence can be removed without affecting its functional integrity during the maturation of uricase.

[0097] In the present embodiment, a specific signal peptide PTS1 is added to the C-terminus of the uricase gene, and the signal peptide can be transported to the peroxisome by binding to the peroxisomal receptor (PEX5) and transporting through the PEX14 membrane pore. In this design, the processing site of the signal peptide is also optimized to ensure that the N-terminal sequence can be removed without affecting its functional integrity during the maturation of uricase.

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

[0099] The 5' homology arm sequence is usually located at the 5' end of the DNA molecule; the 3' homology arm sequence is usually located at the 3' end of the DNA molecule. The 5' homology arm sequence and the 3' homology arm sequence are designed and inserted into the recombinant DNA molecule for homologous recombination.

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

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

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

[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), and the nucleotide sequence of the 3'UTR 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, the recombinant DNA molecule can be produced by: constructing a recombinant plasmid comprising a DNA molecule sequence; digesting the recombinant plasmid with a Type IIS restriction endonuclease or a Type II blunt-end restriction endonuclease to obtain a nucleic acid molecule.

[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-end restriction endonucleases can include at least one of Afe I, Alu I, BsaA I, BstU I, BstZ17 I, DraI, EcoR V, 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 of the embodiments of this specification provides a method for optimizing the uricase gene sequence, which aims to obtain a gene fragment encoding a mutant uricase by modifying a specific site of the uricase gene to improve its stability and biological activity and reduce its immunogenicity. In some embodiments, the gene fragment encoding the mutant uricase can be obtained by the following steps.

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

[0110] Highly conserved positions refer to the positions where the amino acid sequences are highly consistent in different species. The active site refers to the amino acid residues or regions in the uricase molecule that are directly involved in the catalytic reaction. For example, in the uricase of humans and other mammals, the amino acid residues at positions 40 to 50 can serve as conserved catalytic active sites.

[0111] Avoid hydrolase cleavage sites: Hydrolases (e.g., lysosomes) often cleave proteins at certain specific sequences, resulting in loss of enzyme function or instability. Therefore, when optimizing the amino acid fragment of urate oxidase, 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 refer to amino acid residues on the uricase molecule that can covalently bind to ubiquitin molecules. Ubiquitination is an important regulatory mechanism in the process of protein degradation, which can regulate the stability and function of uricase. When predicting ubiquitination sites, it is necessary to evaluate whether these sites will affect the structure of the active site or its conservation. In some embodiments, the ubiquitination sites in uricase may include K9, K41, K54, etc.

[0114] According to the ubiquitination site evaluation results, the amino acid sequence is modified: in response to the predicted ubiquitination site being located at the active site or having a greater impact on conservation, the original amino acid sequence of uricase is retained to avoid the negative effects that may be caused by the mutation; in response to the predicted ubiquitination site not significantly affecting the active site structure or conservation of the enzyme, the thermodynamics, thermal stability, structural changes, hydrogen bond changes, etc. of the uricase after mutation can be predicted, and glycosylation masking is performed based on the amino acid sequence (for example, by introducing glycosylation modification to mask the influence of the ubiquitination site) to reduce the effect of ubiquitination on enzyme function. In some embodiments, glycosylation modification includes 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 of uricase can increase glycosylation modifications at these positions. Glycosylation can increase protein stability and may reduce the adverse effects of ubiquitination by changing the spatial configuration or surface properties of the protein.

[0117] For example, the introduction of a NXT-type glycosylation site at position 137 of urate oxidase may mask the ubiquitination reaction at this site by glycosylation, thereby reducing the degradation effect of ubiquitination on the enzyme.

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

[0119] O-linked glycosylation usually occurs on the serine (Ser) or threonine (Thr) residues of amino acids. This modification can add sugar molecules to the surface of proteins, enhance protein stability and prevent them from being ubiquitinated and degraded. O-linked glycosylation can also affect the structure of proteins, making them more adaptable to the environment in the body.

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

[0121] Sialylation glycosylation modification:

[0122] Sialylation is a common form of glycosylation, especially the addition of sialic acid molecules (such as sialic acid, N-acetylneuraminic acid) at the end of N- or O-glycosylation. This modification can introduce negative charges to proteins, enhance their stability, and reduce recognition by the immune system, thereby extending their half-life in the body.

[0123] In some embodiments, a sialic acid molecule can be added to the N-linked glycosylation-modified end of uricase to enhance the negative charge of the protein, reduce the likelihood of it being recognized by the immune system, and thereby improve its stability in the body.

[0124] Fucosylation:

[0125] Fucose-rich glycosylation can affect protein stability, resistance to degradation, and cell receptor binding. Fucose is a sugar molecule that often appears on the cell surface and in the glycosylation process of some secreted proteins. Introducing fucose-rich modifications can help reduce the degradation of urate oxidase and increase its activity in the body.

[0126] In some embodiments, introducing fucose glycosylation modification (e.g., adding fucose-rich sugar chains) at certain N-linked or O-linked glycosylation sites of uricase can improve its stability in vivo and reduce possible immunogenic reactions.

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

[0128] GalNAc glycosylation modification can be used to change 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 this position, thereby enhancing its stability and reducing the negative effects of ubiquitination.

[0130] Design and optimization of candidate nucleotide sequences: After the optimization of the amino acid sequence is completed, a candidate amino acid sequence of the mutant uricase and a candidate nucleotide sequence (e.g., a candidate RNA sequence) corresponding to the candidate amino acid sequence are obtained by introducing a reasonable glycosylation site. In some embodiments, the RNA sequence can be further optimized. For example, the candidate RNA sequence can be further optimized by MFE value and CAI to ensure its efficient expression in cells.

[0131] The MFE value refers to the free energy of RNA secondary structure and can reflect the most stable conformation of RNA molecules.

[0132] The CAI value refers to the coding fitness index, which is used to measure the degree of optimization of the target gene to improve its translation efficiency in the host cell.

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

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

[0135] The embodiments of this specification provide a highly stable, highly active and low immunogenic mutant uricase and a preparation method thereof. The mutant uricase can be used to treat diseases such as hyperuricemia and gout, and has better clinical application prospects.

[0136] In some embodiments, the recombinant DNA molecule can be a vector. The vector used herein refers to a tool used to carry foreign DNA fragments and perform transcription reactions in cells to produce RNA.

[0137] Vectors are usually circular DNA molecules, such as plasmids or viruses (such as adenovirus, adeno-associated virus, etc.), bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), etc. These vectors have the ability to replicate themselves, can replicate independently in cells, and can also carry exogenous genes, such as protein coding genes, RNA genes, etc.

[0138] In some embodiments, the vector can be designed to contain specific start codons, regulatory elements, and stop codons to allow the exogenous DNA in the cell to be transcribed to produce RNA, which can be protein-encoding mRNA or other non-coding RNA.

[0139] In some embodiments, an in vitro transcription template can be obtained based on the above-mentioned 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 a variety of methods, for example, the in vitro transcription template can be directly obtained 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 a plasmid with a restriction endonuclease to obtain the in vitro transcription template.

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

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

[0143] In some embodiments, mutant uricase can be produced by a circular RNA expression system. Circular RNA has long stability and efficient expression characteristics. By using a circular RNA expression system, the expression efficiency and stability of uricase can be significantly improved.

[0144] One of the embodiments of the present specification provides a method for preparing an RNA molecule, 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 expressing comprises: performing an in vitro transcription reaction on the recombinant DNA molecule to obtain a 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 causing the linear RNA to self-circularize to generate circular RNA.

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

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

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

[0150] In some embodiments, the method further comprises encapsulating the circular RNA with lipid nanoparticles (LNP). LNP encapsulation can enhance the advantages of circular RNA in protein production. LNP is the most advanced nanoparticle carrier that can be used to target specific cells using endogenous or exogenous ligands by encapsulating circular RNA. The endocytosis of LNP destabilizes the endosomal membrane and releases the circular RNA into the cytoplasm. LNP can solve many problems of circular RNA molecules, making them less prone to degradation and promoting cellular uptake.

[0151] In some embodiments, encapsulating circular RNA using LNP further includes: dissolving LNP in ethanol to obtain an LNP solution; dissolving circular RNA in a sodium acetate solution to obtain a circular RNA solution; 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 amine 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 also be used, such as gold nanoparticles (AuNPs), engineered exosomes, but not limited to them.

[0155] The embodiments of this specification provide a method for treating a disease associated with high uric acid levels in a subject, the method comprising administering a pharmaceutically acceptable amount of RNA to the subject.

[0156] As used herein, "subject" refers to a human or an animal. Typically, the animal is a vertebrate, such as a primate (e.g., chimpanzee, cynomolgus monkey, spider monkey, and macaque), a rodent (e.g., mouse, rat, marmot, ferret, rabbit, and hamster), livestock, or prey (e.g., cattle, horses, pigs, deer, bison, buffalo, cats). In some embodiments, the subject is a mammal, such as a primate, such as a human.

[0157] The term "pharmaceutically acceptable amount" as used herein refers to the amount of circRNA that provides a therapeutic benefit when treating a disease with high uric acid levels or reducing uric acid levels, for example, an amount that provides a statistically significant reduction in, for example, serum uric acid. The determination of a pharmaceutically acceptable amount is entirely within the capabilities of those skilled in the art. Typically, a pharmaceutically acceptable amount can vary with the subject's medical history, age, condition, sex, and severity and type of the subject's medical condition and the administration of other pharmaceutically active agents.

[0158] In some embodiments, the circular RNA can be administered to a subject at a dose of 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 is administered every two days, every four days, every week, 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 four days, every two weeks, every four days... or at any time interval within 3 years.

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

[0160] One of the embodiments of this specification provides an application of RNA in the preparation of a drug for reducing uric acid in a subject or treating a disease associated with high uric acid levels.

[0161] The present invention is illustrated by the following examples, which are not intended to be limiting of the present invention. Example Example 1 Optimization of the sequence encoding uricase

[0162] The wild-type sequence of Aspergillus flavus urate oxidase was subjected to amino acid sequence mutations, including the following mutations: C103A, S297K, K299S and K301R (mutant sequences corresponding to SEQ ID NO.1 or SEQ ID NO.10), K5R; K21R; K24R; C104A; K115R; K190R; K290R; S297K; K299S; K301R (mutant sequence corresponding to SEQ ID NO.2) and K5R; K21R; K24R; C104A; K115R; E137N; K139T; K190R; K290R; S297K; K299S; K301R (mutant sequence corresponding to SEQ ID NO.3).

[0163] The wild type of pig (Pig / Sus scrofa) uricase was structurally optimized to obtain the nucleotide sequence shown in SEQ ID NO. 11. The wild type of pig (Pig / Sus scrofa) uricase was subjected to amino acid mutations, including the following mutations: K9R; K54R; K55R; K82R; K85R; K103N; K126R; K272N; T301S (mutation sequence corresponding to SEQ ID NO. 4), K9R; K54R; K55R; K82R; K85R; K103N; K126R; Q201N; R203T; K272N; T301S (mutation sequence corresponding to SEQ ID NO. 5).

[0164] The wild type of baboon (PAPHA) uricase was structurally optimized to obtain the nucleotide sequence shown in SEQ ID NO. 12. The wild type of baboon (PAPHA) uricase was subjected to amino acid mutations, including the following mutations: K9R; K54R; K55R; K82R; K85R; K126R; K272N; K291R (the mutant sequence corresponds to SEQ ID NO. 6). Example 2 Preparation of RNA expressing mutant uricase

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

[0166] In this embodiment, the E2 fragment and the E1 fragment are designed by taking the gene containing the type I intron of the genus Anabaena sp. as an example, but it is not limited to the gene of the genus Anabaena sp., and can also be the E2 and E1 fragments of other sources of the same gene. Between the 5' homology arm sequence A (shown in SEQ ID NO.16) and the 3' homology arm sequence B (shown in SEQ ID NO.17) of the vector, the inserted fragment and the fragment derived from the vector are connected together by sequence recombination, and transformed into competent cells for pressure screening to obtain a plasmid that can express the urate oxidase gene.

[0167] Schematic diagram of the construction of the plasmid expressing the uricase gene Figure 1 As shown, wherein, E2 (shown in SEQ ID NO.7); IRES sequence (shown in SEQ ID NO.9); 5'-terminal signal peptide element (the translated amino acid sequence is shown in any one of SEQ ID NOs.13, 24-28); urate oxidase coding region (Uricase CDS, shown in any one of SEQ ID NOs.1-6, 10-12); 3'-terminal signal peptide element (the translated amino acid sequence is shown in any one of SEQ ID NOs.22-23); terminator (2*TGA); 3'UTR (shown in SEQ ID NO.18); E1 (shown in 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; H 2 0 to 50μL.

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

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

[0173] Prepare 2% DNA agarose gel, perform electrophoresis at 120V for 30min, and use Molpure DNA Purification Kit to recover the PCR product to obtain a 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 order: 2μL of 10×Transcription Buffer; 1.5μL of 100mM ATP / GTP / CTP / UTP mixture; 500ng-1μg of DNA template; 1μL of T7 Enzyme Mix; add nuclease-free water to 20μL. Use a pipette to gently mix (or vortex) the components, and briefly centrifuge to collect the reaction solution to the bottom of the tube. Incubate at 37℃ for 1 hour and 45℃ for 1h.

[0176] 3. DNase I Digestion

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

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

[0179] In some embodiments, the DNA template may further include an intron fragment, and the intron fragment, E1 fragment, and E2 fragment may be from the same genome. For example, the intron fragment, E1 fragment, and E2 fragment may be from the genus Anabaena. When the template DNA contains an intron fragment, after the in vitro transcription reaction is digested with DNase I, 20 μL of 10× cyclization buffer and H 2 0 to 200 μL and incubate at 50-55°C for 20 min to generate circular RNA.

[0180] 4. RNA Purification

[0181] (1) Oligo dT purification

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

[0183] (2) SEC purification

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

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

[0186] The specific steps are as follows:

[0187] (1) Cell transfection

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

[0189] Preparation before transfection: Prepare transfection complex: In a sterile 1.5 mL centrifuge tube, mix Hieff mRNA Transfection Reagent (40809ES03) was mixed with an appropriate amount of Opti-MEM medium (Source Culture, L530KJ), gently mixed, and allowed to stand at room temperature for 5 minutes. In another sterile 1.5 mL 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 (reduced serum medium). Add the prepared transfection complex dropwise to each well of the 6-well plate, and gently shake the culture plate to ensure uniform distribution. After culturing the cells back in a 37°C, 5% CO2 incubator for 4-6 hours, replace with complete medium and continue culturing for 48 hours.

[0193] (2) Cell lysis

[0194] The cell pellet was gently flicked off, and then 150 μL of pre-cooled lysis buffer and PMSF (final concentration of 1 mM) were added to a 6-well plate for lysis. The plate was placed on ice, and the cell suspension was gently blown off. The plate was placed in a 4°C refrigerator or an ice-water mixture for lysis for 15 min.

[0195] (3) Centrifugal treatment

[0196] Centrifugation conditions: speed 12000 rpm, 4°C, time 10 min. Separate the supernatant.

[0197] (4) Protein concentration determination

[0198] The protein concentration was determined using a BCA kit (Adamas life, E8053-100T-PKG).

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

[0200] Sodium borate buffer (1 M, pH 8.5) was used to dissolve uric acid to prepare a 0.6 mM uric acid working solution.

[0201] (6) Urate oxidase activity detection

[0202] At 25°C and pH 8.5, the amount of enzyme that converts 1 μmol of uric acid into allantoin per minute is defined as one activity unit (U). Take 200 μL of 0.6 mM uric acid solution in a test tube, add 20 μL of protein (original concentration, 1 / 5 concentration, 1 / 10 concentration, the experimental groups are numbered H / M / L respectively), and use the uric acid (UA) detection kit (phosphotungstic acid colorimetric method) (LEAGENE, TC1197) to detect the uric acid concentration at 5 min, 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, and 360 min. In addition to the detection at the above time points, the control group was also detected at 0 min. According to the formula U = △UA / T (△UA is the change in uric acid concentration, T is the reaction time), the final result is calculated using the relative enzyme activity (U / mg) to compare the enzyme activity between the groups. The results are as follows Figure 3 As shown, the activity of the pig mutant was increased by 9.1% and 12.5% ​​compared with the wild type. Among them, pWU is the original sequence of pig urate oxidase, pMU1 is the pig urate oxidase mutant corresponding to the mutant sequence SEQ ID NO.4, and pMU2 is the pig urate oxidase mutant corresponding to the mutant sequence SEQ ID NO.5.

[0203] The same method was used to determine that the relative activity of the mutant derived from Aspergillus flavus was 1.82 and 2.95 times higher than that of the original sequence after RNA optimization. Figure 4 , corresponding to optimized sequence 1, corresponding to optimized sequence 2), the enzyme activity of the mutant after the introduction of the NxT site decreased slightly by 20±5% (not shown in the figure).

[0204] The same method was used to determine that the activity of the baboon-derived optimized sequence was 1.68 times higher than that of the original sequence, and the activity of the deubiquitinating mutant was 1.36 times higher. (Not shown in the figure) Example 4 Long-term effect and stability test of uricase mutants

[0205] The uricase with optimized RNA structure 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. Some samples were taken out and the activity of uricase was determined by referring to the method in Example 3. The activity and enzyme activity retention rate were compared. The results are shown in FIG. Figure 4 As shown. Figure 4 It can be seen that the optimized sequence 1 (corresponding to SEQ ID NO. 1) and the optimized sequence 2 (corresponding to SEQ ID NO. 10) still have significant activity after 144 hours. Example 5 Production Detection of Urate Oxidase Mutants

[0206] Take an appropriate amount of lysed protein sample (usually 20-30 μg, the specific amount is adjusted according to experimental requirements), add an equal volume of 5X SDS-PAGE protein loading buffer (Biyuntian, P0015B), and mix gently. Make sure that the protein and loading buffer are fully mixed to avoid abnormal bands during electrophoresis.

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

[0208] Centrifuge briefly (about 10,000 rpm, 30 seconds) to collect droplets on the tube wall and ensure that the sample is completely concentrated at the bottom of the tube.

[0209] Electrophoresis detection: Prepare SDS-PAGE gel (select the appropriate gel concentration according to the molecular weight of the target protein, such as 10% or 12% separation gel). Add the prepared protein sample to the gel loading well, and add prestained protein molecular weight markers (such as 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 in the figure, the protein production of the porcine urate oxidase mutants was increased. By comparing the electrophoresis band intensity or Western blot signal intensity, the changes in protein expression levels can be quantitatively analyzed.

[0211] Detected in the same manner, the yield of the Aspergillus flavus-derived stable mutant was also improved (not shown in the figure).

[0212] According to the experimental results, the enzyme activity of the pig-derived mutant was significantly improved compared with the wild type (the increase was 9.1% and 12.5%, respectively). Combined with the phenomenon of increased protein production, it is speculated that the increase in activity may be related to the optimization of protein expression efficiency, that is, the enzyme activity per unit protein (specific enzyme activity) increases synchronously due to the increase in expression. After the RNA structure of the aflatoxin-derived urate oxidase was optimized, the protein expression efficiency was significantly increased, and the activity decayed at a lower rate over time. The mutant showed a contradictory trend: although the protein production was significantly increased, its enzyme activity was lower than that of the original mutant (a decrease of 20±5%). This shows that the introduction of glycosylation sites may weaken the catalytic efficiency of the enzyme through conformational changes or active site interference-although the increase in protein production can increase the total enzyme amount to a certain extent, the loss of unit enzyme activity has not been fully compensated, which ultimately leads to a decrease in overall activity. It is worth noting that the deubiquitination, introduction of glycosylation and increased expression characteristics of the Aspergillus flavus mutant may give it a longer-lasting protein retention time in the body. This advantage may make up for the lack of enzyme activity in vitro, thereby reflecting potential value in application scenarios such as long-term drug efficacy. Example 6 In vivo experiment

[0213] Experimental subjects: Uox-KO mice, male, 4-5 weeks old (purchased from Biocytogen), were bred in the laboratory for 7 days and injected according to group numbers.

[0214] Injection method: The circular RNA (from Aspergillus flavus, containing C103A, S297K, K299S and K301R mutations, and optimized RNA structure, i.e. corresponding to SEQ ID NO.1) was encapsulated by a lipid nanoparticle (LNP) delivery system and injected through the tail vein of mice.

[0215] Blood collection method: orbital blood collection is performed once before injection, once every 3 days, and subsequently, eye blood is collected every 7 days.

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

[0217] The results are as follows Figure 6 As shown, the circular RNA expressing urate oxidase has a very strong pharmacological effect in mice in the first 7 days, followed by a very long drug maintenance period (21 days).

[0218] As described above, the basic concepts have been described, and it may be quite clear to those skilled in the art after reading this detailed disclosure, which is intended to be presented by way of example only and not by way of limitation. Although not expressly stated herein, various changes, improvements and modifications may be made by those skilled in the art. Such changes, improvements and modifications are intended to be proposed by the present disclosure and are within the spirit and scope of the exemplary embodiments of the present disclosure.

[0219] In addition, certain terms have been used to describe embodiments of the present disclosure. For example, the terms "one embodiment," "embodiment," and / or "some embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, it is emphasized and should be understood that two or more references to "embodiment" or "one embodiment" or "alternative embodiment" in various parts of the present disclosure do not necessarily all refer to the same embodiment. In addition, particular features, structures, or characteristics may be appropriately combined in one or more embodiments of the present disclosure.

[0220] Furthermore, the order in which processing elements or sequences are recited, or the use of numbers, letters, or other designations, is not intended to limit the claimed processes and methods to any order other than that which may be specified in the claims.

[0221] In some embodiments, the numbers representing quantities, characteristics, etc. used to describe and claim certain embodiments of the present application should be understood to be modified by the terms "about", "approximately" or "substantially" in some cases. For example, "about", "approximately" or "substantially" may represent a ±20% variation of the value it describes, unless otherwise stated. Therefore, in some embodiments, the numerical parameters listed in the written description and the attached claims are approximate values, which may vary depending on the desired characteristics desired to be obtained in a particular embodiment. In some embodiments, the numerical parameters should be interpreted according to the number of reported significant digits and applying ordinary rounding techniques. Although the numerical ranges and parameters setting forth the broad scope of some embodiments of the present application are approximate, the numerical values ​​listed in the specific examples are reported as accurately as possible.

[0222] Each patent, patent application, patent application publication, and other material (e.g., articles, books, specifications, publications, documents, things, and / or the like) cited herein is hereby incorporated by reference in its entirety for all purposes, except any litigation document history related thereto, anything that is inconsistent or conflicting with this document, or anything that may have the effect of limiting the broadest scope of the claims now or later related to this document. For example, if there is any inconsistency or conflict between the description, definition, and / or use of terminology related to any incorporated material and the description, definition, and / or use of terminology related to this document, the description, definition, and / or use of terminology in this document shall control.

[0223] Finally, it should be understood that the embodiments of the present application disclosed herein are only used to illustrate the principles of the embodiments of the present application. Other modifications that may be adopted may be within the scope of the present application. Therefore, as an example, but not limited to, alternative configurations of the embodiments of the present application may be utilized according to the teachings of this article. Therefore, the embodiments of the present 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 operably linked and arranged in the following order in the 5' to 3' direction: (a) Exon E2 fragment; (b) internal ribosome entry site (IRES) fragment; (c) 5' signal peptide element; (d) a gene fragment encoding urate oxidase; (e) a 3' signal peptide element; and (f) Exon E1 fragment; wherein the gene fragment encoding uricase comprises any one of SEQ ID NOs. 1-6, 10-12.

2. The recombinant DNA molecule according to claim 1, characterized in that The gene fragment encoding uricase oxidase comprises at least one of a mutant uricase oxidase coding sequence derived from Aspergillus flavus, a mutant uricase oxidase coding sequence derived from pig and a mutant uricase oxidase coding sequence derived from baboon; the amino acid mutation site of the mutant uricase oxidase derived from Aspergillus flavus comprises at least one of K5R, K21R, K24S, K49S, K139Q, C104A, K115R, E137N, K139T, K190R, K204R, K218R, K290R, S297K, K299S and K301R; the amino acid mutation site of the mutant uricase derived from pig comprises at least one of The points 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 mutant urate oxidase from baboon include at least one of K9R, K54R, K55R, K82R, K85R, K126R, K272N, and K291R.

3. The recombinant DNA molecule according to claim 1, characterized in that The gene fragment encoding uricase oxidase is structurally optimized, and the gene fragment encoding uricase oxidase in the optimized DNA molecule includes any one of SEQ ID NOs. 1, 10-12.

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

8.

5. The recombinant DNA molecule according to claim 1, wherein the IRES fragment is derived from Taura syndrome virus, blood-sucking assassin bug virus, Reye's encephalomyelitis virus, simian virus 40, fire ant virus 1, graminearum aphid virus, reticuloendotheliosis virus, Forman polio virus 1, soybean looper virus, Kashmir bee virus, human rhinovirus 2, green leafhopper virus-1, human immunodeficiency virus type 1, green leafhopper virus-1, lice P virus, hepatitis C virus Viruses, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid-like virus, encephalomyocarditis virus (EMCV), fruit fly C virus, crucifer 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 ringspot virus, swine fever virus, human FGF2, human SFTPA1, human AML1 / RUN X1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1a, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, salivirus, coxsackievirus, double 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 picornavirus, turnip shrivelled disease virus, aptamer for eIF4G, coxsackievirus A (CVB1 / 2), or coxsackievirus B3 (CVB3). The recombinant DNA molecule according to claim 1 , wherein the IRES fragment comprises SEQ ID NO.

9.

7. The recombinant DNA molecule according to claim 1, characterized in that The 5'-end signal peptide encoded by the 5'-end signal peptide element is used to guide the uricase oxidase to enter the endoplasmic reticulum to complete the glycosylation modification, and the 3'-end signal peptide encoded by the 3'-end signal peptide element is used to transport the glycosylated uricase from the endoplasmic reticulum to the peroxisome. The amino acid sequence translated by the 5'-end signal peptide element includes any one of SEQ ID NOs.13, 24-28, and the amino acid sequence translated by the 3'-end signal peptide element includes any one of SEQ ID NOs.22-23.

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

17.

9. The recombinant DNA molecule according to claim 6, characterized in that The 5'-end signal peptide element is preceded by a start codon, and the 3'-end signal peptide element is followed by two stop codons.

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

18.

11. An RNA molecule, characterized in that The RNA is produced based on the recombinant DNA molecule according to any one of claims 1 to 10, and the RNA comprises linear RNA or circular RNA.

12. A method for preparing an RNA molecule, characterized in that: The method comprises expressing a recombinant DNA molecule according to any one of claims 1 to 10 in a host cell or a cell-free expression system.

13. Use of the RNA according to claim 11 in the preparation of a drug for reducing uric acid in a subject or treating a disease associated with high uric acid levels.

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