Glucagon fusion protein, polynucleotide and application

Abstract

The invention provides a glucagon fusion protein, polynucleotide and application, and relates to the technical field of biology. The glucagon fusion protein comprises the following structure: [GCG]-[(L)-(GCG)] m-[X] n, gCG is a glucagon fragment, and L is a connecting peptide; x is a fragment with an amino acid sequence as shown in SEQ ID No.2; m is an integer greater than or equal to 0, n is 0 or 1, and m and n are not 0 at the same time. According to the glucagon fusion protein, the problem that glucagon cannot be normally expressed on the protein level is successfully solved by adding a carbon terminal extension or short peptide concatemer, and a new thought is provided for more effectively regulating related metabolic pathways.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a glucagon fusion protein, a polynucleotide, and its applications. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Glucagon (GCG) is a peptide hormone secreted by pancreatic A cells that regulates blood glucose levels and lipid metabolism. As an insulin antagonist, GCG plays a crucial role in maintaining glucose homeostasis in animals and humans. To increase blood glucose, GCG works synergistically through multiple mechanisms to promote hepatic glucose output, increase glycogenolysis and gluconeogenesis, while coordinating glycemic production by reducing glycogen synthesis and glycolysis. Compared to healthy subjects, diabetic patients and animals exhibit abnormalities not only in insulin secretion but also in GCG secretion. Hyperinsulinemia and altered insulin / GCG ratios play a key role in initiating and maintaining pathological hyperglycemia. Therefore, GCG and its receptor have been extensively explored in recent years as potential targets for diabetes treatment.

[0004] Currently, GCGR agonists are often used in combination with GLP-1R agonists, usually in peptide form for direct treatment, but they suffer from a short half-life. Considering the higher expression levels and longer half-life of RNA, we explored a novel approach: RNA-mediated protein replacement therapy. However, GCG is a short peptide composed of 29 amino acids, and its encoding sequence has a low molecular weight, making its expression at the protein level challenging.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a glucagon fusion protein and its polynucleotides to solve the problem of glucagon expression difficulties. Based on this glucagon fusion protein, this invention also provides related vectors, cells, preparation methods, and their applications.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] In a first aspect, a glucagon fusion protein is provided, comprising the following structure:

[0009] Equation (Ⅰ): [GCG]-[(L)-(GCG)] m -[X] n ;

[0010] GCG stands for glucagon fragment, and L stands for linker peptide;

[0011] X is the amino acid sequence shown in SEQ ID No. 2;

[0012] m is an integer ≥ 0, n is 0 or 1, and m and n are not both 0.

[0013] Secondly, a polynucleotide is also provided, which contains a CDS encoding a structure of formula (II) or formula (III):

[0014] Formula (II): [Y]-[GCG]-[(L1)-(GCG)] m -[X] n ;or,

[0015] Formula (III): [Y]-[GCG]-[(L2)-(Y)-(GCG)] m -[X] n ;

[0016] Y is the signal peptide, GCG is the glucagon fragment, L1 is the flexible linker peptide, L2 is the cleavable linker peptide, and X is the amino acid sequence shown in SEQ ID No. 2.

[0017] m is an integer ≥ 0, n is 0 or 1, and m and n are not both 0.

[0018] Thirdly, a vector is also provided that carries the polynucleotide of the second aspect.

[0019] Fourthly, a cell is also provided, the cell surface of which the glucagon fusion protein of the first aspect is carried, or the polynucleotide of the second aspect is carried, or the carrier of the third aspect is contained.

[0020] Fifthly, a method for preparing a glucagon fusion protein is also provided, the method comprising expressing the glucagon fusion protein in cells.

[0021] The sixth aspect also provides the use of the glucagon fusion protein of the first aspect, or the polynucleotide of the second aspect, or the carrier of the third aspect, or the cell of the fourth aspect, or the preparation method of the fifth aspect in the preparation of a pharmaceutical composition for the prevention and / or treatment of metabolic disorders.

[0022] In a seventh aspect, a medicament is also provided, comprising the glucagon fusion protein of the first aspect, or the polynucleotide of the second aspect, or the carrier of the third aspect, or the cell of the fourth aspect, the medicament composition being used for the prevention and / or treatment of metabolic disorders.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention successfully overcomes the problem of poor expression of wild-type glucagon by adding signal peptides, C-terminal extensions, or short peptide tandems, alleviating the limitation that bio-fermentation methods cannot achieve normal expression of glucagon at the protein level, and providing a new approach for more effectively regulating related metabolic pathways.

[0025] In this preferred approach, glucagon analogs are encoded via mRNA and circRNA, overcoming numerous limitations of traditional recombinant protein therapy methods, such as short half-life, instability, immunogenicity, and the inability to replace intracellular proteins. Utilizing advanced mRNA and circRNA expression technologies, sustained expression of glucagon has been successfully achieved, extending its half-life and providing a new pathway for its stable expression.

[0026] This invention provides an innovative approach for the regulation of related metabolic pathways and the treatment of related diseases. The effects of glucagon can occur and subside rapidly within minutes. In a preferred embodiment, glucagon fusion protein is encoded in vivo using mRNA and circRNA expression technology to simulate the pulsatile secretion of glucagon from the pancreatic islets under physiological conditions. This pulsatile release of glucagon more effectively induces hepatic glucose output both in vitro and in vivo. Detailed Implementation

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

[0028] As used herein, the terms “comprising,” “containing,” “including,” “comprising,” and “having” are defined as encompassing (i.e., open-ended expressions). “Comprising,” “containing,” “including,” “comprising,” and “having,” or any other variations thereof, are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but may also include other elements not expressly listed or inherent to those elements. An element preceded by “comprising…one (a)” does not exclude (without further limitation) the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0029] The terms "variant" and "analyte" used herein are used interchangeably and refer to biological molecules that have been modified or altered, preferably retaining their biological activity. For example, "variant" and "analyte" of a polypeptide refer to a polypeptide whose amino acid sequence is different but whose function is not changed or is not significantly altered by one or more substitutions, deletions, insertions, fusions, truncations, or any combination thereof.

[0030] In this article, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified forms. Common naturally occurring amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In this article, the Chinese names, three-letter abbreviations, and single-letter abbreviations of amino acids can be used interchangeably.

[0031] The term "glucagon" as used herein refers to a polypeptide corresponding to amino acids 53 to 81 of the proglucagon, hereinafter also referred to as "GCG". Examples include wild-type glucagon having the amino acid sequence shown in SEQ ID No. 1, or variants of wild-type glucagon shown in SEQ ID No. 1 that substantially retain glucagon receptor agonist activity.

[0032] In this article, the term "glucagon receptor agonist" refers to a substance that has glucagon receptor agonist activity, such as wild-type glucagon or analogues of wild-type glucagon.

[0033] The “Exendin4” mentioned in this article refers to the polypeptide found in the salivary secretions of the venomous lizard (J. Biol. Chem, 265, 20259 20262, 1990; J. Biol. Chem, 267, 74027405, 1992) and its variants that substantially retain the GLP1 receptor agonist function. Wild-type Exendin4 is highly homologous (53%) to GLP1 (736), with the sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS, SEQ ID No. 16).

[0034] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. The polynucleotide encodes the aforementioned glucagon fusion protein, optionally encoding either the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0035] In this article, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the cells.

[0036] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0037] The terms “cell,” “cell line,” and “cell culture” used in this article are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, and may differ from primary cells morphologically and / or in genomic DNA.

[0038] As used herein, the term "pharmaceutical composition" refers to a combination of at least one drug substance and optionally a pharmaceutically acceptable carrier or excipient, combined together to achieve a particular purpose. In some embodiments, the pharmaceutical composition comprises combinations that are separate in time and / or space, provided that they can work together to achieve the objectives of the invention.

[0039] The "signal peptide" described herein is a short peptide located at the N-terminus of a protein that guides the transmembrane translocation of newly synthesized polypeptide chains to the endoplasmic reticulum, thereby entering the secretory pathway. After being cleaved by a signal peptidase, the mature protein is released, while the signal peptide is degraded. In a first aspect, a glucagon fusion protein is provided, comprising the following structure:

[0040] Equation (Ⅰ): [GCG]-[(L)-(GCG)] m -[X] n The structure is represented in the direction from the N end to the C end.

[0041] GCG stands for glucagon fragment, and L stands for linker peptide;

[0042] m represents the number of repeats of [(L)-(GCG)], where m is an integer ≥ 0, for example, it can be, but is not limited to, 1, 2, 3, 4 or 5; n is the number of repeats of [X], where n is 0 or 1, and m and n are not both 0, that is, when the glucagon fusion protein does not have the GCG repeat fragment, it contains the extended fragment X.

[0043] The amino acid sequences of each glucagon fragment can be the same or different, and the differences include partial differences or all glucagon fragments having different amino acid sequences.

[0044] In an optional embodiment, the amino acid sequence of at least one GCG is as shown in SEQ ID No. 1, and preferably the amino acid sequence of each GCG is as shown in SEQ ID No. 1.

[0045] In an optional embodiment, the glucagon fusion protein contains two or more linking peptides, which may or may not be the same.

[0046] In an optional implementation, the linker peptide is a flexible linker peptide.

[0047] In an optional embodiment, the amino acid sequence of the flexible linker peptide is (GGGGS). x x = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably x = 1.

[0048] X is an amino acid sequence as shown in SEQ ID No. 2, which originates from the N-terminus of Exendin 4 and is amino acid 29 to 39 of SEQ ID No. 16.

[0049] In an optional embodiment, the glucagon fusion protein comprises any of the structures of formulas (i) to (iii):

[0050] Formula (i): [GCG]-[X];

[0051] In an optional embodiment, the amino acid sequence of GCG in formula (i) is shown in SEQ ID No. 1.

[0052] Formula (ii): [GCG]-[(L)-(GCG)];

[0053] In an optional embodiment, the amino acid sequence of each GCG in formula (ii) is as shown in SEQ ID No. 1.

[0054] In an optional embodiment, L in formula (ii) is GGGGS, and the amino acid sequence can be selected as shown in SEQ ID No. 5.

[0055] Formula (ⅲ): [GCG]-[(L)-(GCG)]-[X];

[0056] In an optional embodiment, the amino acid sequence of each GCG in formula (iii) is as shown in SEQ ID No. 1.

[0057] In an optional embodiment, L in formula (ii) is GGGGS, and the amino acid sequence can be selected as shown in SEQ ID No. 5.

[0058] In an optional embodiment, the amino acid sequence of the glucagon fusion protein includes the sequence shown in SEQ ID No. 39, 40 or 41.

[0059] SEQ ID No. 39:

[0060] HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGPSSGAPPPS.

[0061] SEQ ID No. 40:

[0062] HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGGGSHSQGTFTSDYSKYLDSRRAQDFVQWL MNT.

[0063] SEQ ID No. 41:

[0064] HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGGGSHSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGPSSGAPPPS.

[0065] The glucagon fusion protein in the above embodiments, with the addition of GCG to the C-terminus and / or containing a GCG short peptide tandem, successfully overexpresses GCG analogs, alleviates the limitation of GCG sequence length, and has high agonistic activity for GCG receptors.

[0066] Secondly, a polynucleotide is also provided, which contains a CDS encoding a structure of formula (II) or formula (III):

[0067] Formula (II): [Y]-[GCG]-[(L1)-(GCG)]m-[X]n; or,

[0068] Formula (III): [Y]-[GCG]-[(L2)-(Y)-(GCG)]m-[X]n;

[0069] Y is the signal peptide, GCG is the glucagon fragment, L1 is the flexible linker peptide, L2 is the cleavable linker peptide, and X is the amino acid sequence of the fragment shown in SEQ ID No. 2.

[0070] m represents the number of repeats of [(L1)-(GCG)] or [(L2)-(Y)-(GCG)], where m is an integer ≥ 0, for example, it can be, but is not limited to, 1, 2, 3, 4 or 5; n is the number of repeats of [X], where n is 0 or 1, and m and n are not both 0, that is, when the glucagon fusion protein does not have the GCG repeat fragment, it contains the extended fragment X.

[0071] In an optional implementation, when m≥1, each polynucleotide encoding GCG can be the same or different.

[0072] In an optional implementation, n is 0 in equation (Ⅲ) and does not contain the extended segment X.

[0073] In an optional embodiment, the amino acid sequence of the flexible linker peptide L1 is (GGGGS)x, where x is a positive integer ≥1; optionally, x = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably x = 1.

[0074] In an optional embodiment, the signal peptide Y is a proinsulin signal peptide or a proglucagon signal peptide. The amino acid sequence of the proglucagon signal peptide may optionally be as shown in SEQ ID No. 3.

[0075] In an optional embodiment, the polynucleotide includes DNA.

[0076] In an optional implementation, the DNA sequence encoding GCG is shown in SEQ ID NO. 24 or 25.

[0077] In an optional embodiment, the DNA sequence encoding the proinsulin signal peptide is shown in SEQ ID NO.26.

[0078] In an optional embodiment, the DNA sequence encoding the linker peptide L1 is shown in SEQ ID NO.27.

[0079] In an optional implementation, the DNA sequence encoding X is shown in SEQ ID NO.28.

[0080] In an optional embodiment, the polynucleotide is DNA, and the nucleotide sequence is as shown in SEQ ID NO. 21 or 23.

[0081] In an optional implementation, the polynucleotide includes RNA.

[0082] In an optional implementation, the RNA sequence encoding GCG is shown in SEQ ID NO.29 or 30.

[0083] In an optional embodiment, the RNA sequence encoding the pre-proinsulin signal peptide is shown in SEQ ID NO.31.

[0084] In an optional embodiment, the RNA sequence encoding the linker peptide L1 is shown in SEQ ID NO.32.

[0085] In an optional implementation, the RNA sequence encoding X is shown in SEQ ID NO.33.

[0086] In an optional implementation, the RNA includes mRNA or circular RNA.

[0087] In an optional embodiment, the nucleotide sequence of the CDS of the mRNA or circular RNA is independently shown as SEQ ID NO. 15 or 18.

[0088] In an optional embodiment, the structure of the mRNA includes: 5'UTR-Kozak-CDS-3'UTR-polyA;

[0089] In an optional implementation, the nucleotide sequence of the 5'UTR is shown in SEQ ID NO.34.

[0090] In an optional implementation, the nucleotide sequence of Kozak is GCCACC.

[0091] In an optional implementation, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.36.

[0092] In an optional implementation, the nucleotide sequence of polyA is shown in SEQ ID NO.35.

[0093] In an optional embodiment, the polynucleotide is mRNA, and the nucleotide sequence is as shown in SEQ ID NO.15 or 18.

[0094] In an optional embodiment, the structure of the circular RNA includes: E2 exon-5' spacer region-internal ribosome entry site-Kozak-CDS-3' spacer region-E1 exon;

[0095] In an optional implementation, the nucleotide sequence of exon E2 is shown in SEQ ID NO.19;

[0096] In an optional embodiment, the nucleotide sequence of the 5' spacer region is shown in SEQ ID NO.37;

[0097] In an optional embodiment, the nucleotide sequence of the internal ribosome entry site is shown in SEQ ID NO.14;

[0098] In an optional embodiment, the nucleotide sequence of the 3' spacer region is shown in SEQ ID NO.38;

[0099] In an optional implementation, the nucleotide sequence of the E1 exon is shown in SEQ ID NO.20.

[0100] Thirdly, a vector is also provided that carries the polynucleotide of the second aspect.

[0101] Fourthly, a cell is also provided, the cell surface of which the glucagon fusion protein of the first aspect is carried, or the polynucleotide of the second aspect is carried, or the carrier of the third aspect is contained.

[0102] In an optional implementation, the cells include mammalian cells.

[0103] In an optional embodiment, the mammalian cells include HEK cells (human embryonic kidney cells).

[0104] Fifthly, a method for preparing a glucagon fusion protein is also provided, the method comprising expressing the glucagon fusion protein in cells.

[0105] In an optional implementation, the cells include those described in the fourth aspect.

[0106] In an optional embodiment, the preparation method includes introducing a second-aspect polynucleotide into the cells.

[0107] In an optional embodiment, the RNA is mRNA or circular RNA. Using mRNA or circular RNA to encode glucagon fusion proteins overcomes many limitations of traditional recombinant protein therapy methods, such as the short half-life, instability, and immunogenicity of proteins, as well as their inability to replace intracellular proteins.

[0108] The widespread application of mRNA in the medical field demonstrates its enormous potential. As a key component of gene therapy, mRNA encodes proteins and triggers transient protein expression within cells. Compared to DNA gene therapy, mRNA is not integrated into the genome, resulting only in transient protein expression, thus offering higher safety. Furthermore, mRNA can be produced in cell-free systems via in vitro transcription (IVT), a relatively low-cost process that can be rapidly applied to various therapeutic areas. Through sequence optimization, mRNA can overcome many limitations of traditional recombinant protein therapies, such as the short half-life, instability, and immunogenicity of proteins, as well as their inability to replace intracellular proteins.

[0109] Compared to unmodified mRNA, circular RNA typically exhibits higher stability and lower immunogenicity after purification. The circular structure of circular RNA allows ribosomes to re-engage after one translation cycle, resulting in unique rolling circular translation and improved translation efficiency. Furthermore, in some alternative implementations, insertion into the IRES element or m6A modification of the 5'UTR sequence can further enhance translation efficiency. All these properties endow circular RNA with great potential for producing therapeutic proteins, applicable to protein replacement therapies and vaccine development.

[0110] Therefore, in the preferred embodiment, the preparation of glucagon fusion protein using mRNA and circular RNA is an innovative and efficient solution that is expected to drive progress in the treatment of metabolic diseases.

[0111] In an optional embodiment, the polynucleotide is mRNA, and the preparation method further includes in vitro transcription to prepare mRNA.

[0112] In an optional embodiment, the polynucleotide is a circular RNA, and the preparation method further includes forming a circular RNA through type I introns. Exemplary type I introns include type I introns of cyanobacteria.

[0113] The sixth aspect also provides the application of the glucagon fusion protein of the first aspect, or the polynucleotide of the second aspect, or the carrier of the third aspect, or the cell of the fourth aspect, or the preparation method of the fifth aspect in the preparation of the pharmaceutical composition.

[0114] In a seventh aspect, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the glucagon fusion protein of the first aspect, or the polynucleotide of the second aspect, or the carrier of the third aspect, or the cell of the fourth aspect.

[0115] The pharmaceutical compositions of the sixth or seventh aspect are intended for use on, but are not limited to, cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, guinea pigs, hamsters, dogs, cats, rabbits, camels, donkeys, deer, or humans.

[0116] The pharmaceutical composition of the sixth or seventh aspect is used for the prevention and / or treatment of metabolic disorders, including but not limited to hypoglycemia, type 1 diabetes, kidney disease caused by metabolic disorders, fatty liver, or obesity.

[0117] The pharmaceutical composition of the sixth or seventh aspect is used for the prevention and / or treatment of hypoglycemia. The pharmaceutical composition may be administered via any suitable route, such as oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration.

[0118] In an optional embodiment, the pharmaceutical composition contains a second polynucleotide, which preferably includes mRNA and / or circular RNA, more preferably circular RNA.

[0119] In an optional embodiment, the pharmaceutical composition contains mRNA and / or circular RNA with a nucleotide sequence of CDS as shown in SEQ ID NO. 15 or 18.

[0120] In an optional embodiment, the pharmaceutical composition may further include one or more active pharmaceutical ingredients having other therapeutic effects.

[0121] In an optional embodiment, the pharmaceutical composition further includes a GLP-1 (human glucagon-like peptide-1) receptor agonist and / or a GIP (glucose-dependent insulinotropic peptide) receptor agonist for the treatment of obesity and non-alcoholic fatty liver disease.

[0122] In optional embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient. The acceptable carrier and pharmaceutically acceptable excipient may be any conventional carrier and / or excipient known in the art. These include, but are not limited to, solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, anti-caking agents, flavoring agents, antibacterial agents, suspending agents, coating agents, film-forming agents, fragrances, thickeners, anti-adhesion agents, antioxidants, antioxidant synergists, chelating agents, pH adjusters, adsorbents, plasticizers, surfactants, thickeners, inclusion agents, protectants, humectants, softeners, absorbents, diluents, release regulators, pressure-sensitive adhesives, hardeners, empty capsules, matrix, and drug carrier materials, one or more of these.

[0123] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0124] Example 1: Expression of recombinant plasmids

[0125] Construct GCG receptor agonists. The amino acid sequences of the domains are shown in Table 1, the DNA sequences encoding each domain are shown in Table 3, the amino acid sequences of the CDS-encoded fragments are shown in Table 2, and the DNA sequences are shown in Table 4.

[0126] Table 1. Amino acid sequences of major structural domains

[0127]

[0128]

[0129] Table 2. Amino acid sequences of CDS-encoded fragments

[0130]

[0131] Table 3. DNA sequences of major structural domains

[0132]

[0133] Table 4. DNA Sequences of CDS

[0134]

[0135] The "Structure" column in Tables 2 and 4 indicates the domain composition of the fragment encoded by the CDS. The amino acid sequences of each domain in this structure are shown in Table 1, and the corresponding nucleotide sequences encoding this domain are shown in Table 3. The "abbreviations" are used to indicate the order of the domains from the N-terminus to the C-terminus of the GCG receptor agonist. For example, the abbreviation of SEQ ID No. 10 is 3-1-5-1, indicating that the domain composition of the target fragment encoded by the CDS is proinsulin signal peptide (SEQ ID No. 3)-GCG (SEQ ID No. 1)-G4S linker (SEQ ID No. 5)-GCG (SEQ ID No. 1). The amino acid sequence corresponding to the nucleotide sequence of the CDS is shown in SEQ ID No. 10.

[0136] (1) Gene synthesis: Suzhou Nearshore Protein Technology Co., Ltd. (hereinafter referred to as "Nearshore Protein") performs codon optimization on the gene of GCG fusion protein (amino acid sequence shown in Table 2), adds a signal peptide sequence and an enzyme cleavage site to the optimized DNA sequence, and performs full gene synthesis of the DNA sequence.

[0137] (2) PCR amplification reaction: Primers were synthesized according to the DNA sequence, and NcoI and XbaI restriction sites were added to the primer design. PCR amplification reaction was carried out. PCR amplification was identified by 1.5% agarose gel electrophoresis and imaged using a gel imaging system.

[0138] (3) Cloning and identification of the target gene: After purifying the PCR product, the purified PCR product and the circular plasmid PCMV7B were digested with NcoI and XbaI, respectively. Then, the purified PCR product (i.e., the target gene) and the linear plasmid PCMV7 were separated and recovered by agarose gel electrophoresis. The target gene and the linear plasmid PCMV7 were mixed in a certain ratio for ligation reaction. 5 μL of the ligation product was transformed into competent DH5α cells and cultured overnight. Single colonies were randomly selected, cultured, and plasmid DNA was extracted. The plasmid was then screened and identified by double digestion with NcoI and XbaI. The plasmid PCMV7B-GCG with correct cloning and a high supercoil ratio was obtained. The above experiments were performed by nearshore proteins.

[0139] (4) Expression of plasmid PCMV7-GCG at the cellular level:

[0140] Cell culture: 293T cells were cultured in culture dishes using DMEM medium. When the 293T cells grew to 70-90%, the cells were digested with trypsin, dispersed into single cells, and a cell suspension was prepared with a density of 2.5 × 10 cells / mL. 500 μL of the cell suspension was added to a 24-well plate and incubated overnight.

[0141] Preparation of transfection premix: First, add 25 μL of Opti-MEM to the centrifuge tube. TM Add 1 μL of lipo3000 transfection reagent dropwise to the culture medium, mix well by pipetting, and prepare a liposome premix. Add 25 μL of Opti-MEM to the centrifuge tube. TM In the culture medium, slowly add 1 μg of plasmid PCMV7B-GCG and 1 μL of transfection reagent Lipo 3000, mix well by pipetting, and prepare a DNA premix. Add the liposome premix to the DNA premix, mix well by pipetting, and prepare a DNA-liposome complex, totaling 50 μL. Mix well by pipetting and let stand for 10 min.

[0142] Transfection: Slowly add 50 μL of DNA-liposome complex into the cell suspension, mix gently, and incubate at 37°C for 48 h.

[0143] Collect extracellular supernatant: Centrifuge the transfected cell supernatant at 1000 rpm for 5 min, discard cell debris, and obtain extracellular supernatant.

[0144] Collect the lysis supernatant: Wash the transfected cells three times with pre-cooled PBS, centrifuge briefly and aspirate the PBS, add 120 μL of lysis buffer, lyse for 5 min, and centrifuge at 1000 rpm for 5 min to obtain the intracellular lysis supernatant.

[0145] The detection was performed using the Glucagon ELISA kit (Lianke Biotechnology).

[0146] Standard redissolution: Add a measured amount of pure water according to the label on the bottle to redissolve to 10000 pg / mL;

[0147] Standard dilution: The standards were serially diluted using cell culture medium, with 8 2-fold dilutions (5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312.5 pg / mL, 156.25 pg / mL, 78.13 pg / mL, 0 pg / mL).

[0148] Standard sample addition: 100 μL / well is added to the microplate.

[0149] The negative control was untransfected 293T cells.

[0150] Add sample: Add 100 μL of extracellular supernatant or lysis supernatant to the microplate, cover with sealing film, and incubate at 25°C and 200 rpm for 2 h;

[0151] Add detection antibody: Dilute the detection antibody 1:100 with 1× detection buffer, add 100 μL / well to the microplate; cover with sealing film, incubate at 25℃ and 200 rpm for 1 h;

[0152] Add streptavidin for incubation: Dilute streptavidin 1× detection buffer at a ratio of 1:100 and add 100 μL / well to the microplate; cover with sealing film and incubate at 25℃ and 200 rpm for 45 min.

[0153] Color development: Add 100 μL / well Substrate TMB, incubate at room temperature in the dark for about 10 min, then add 100 μL / well StopSolution to terminate the process. Immediately place the plate in a microplate reader to read the OD value.

[0154] Table 5 shows the OD values ​​detected by a microplate reader after cell transfection with plasmid PCMV7-GCG. Table 6 shows the protein expression levels of plasmid PCMV7-GCG.

[0155] Table 5. OD values ​​of plasmid PCMV7-GCG

[0156] Serial Number (SEQ ID) extracellular supernatant OD value OD value of lysis supernatant 7 0.3554 0.0544 8 0.7778 0.2206 9 0.0683 0.0552 10 1.2494 1.2362 11 0.6742 0.1302 12 0.728 0.2268 13 1.3011 1.2821 negative control 0.0526 0.0565

[0157] Table 6. Protein expression levels of plasmid PCMV7-GCG

[0158] Serial Number (SEQ ID) Extracellular supernatant (pg / mL) Lysis supernatant (pg / mL) 7 934.1 N / A 8 2589.4 406.0 9 N / A N / A 10 >5000 >5000 11 2183.4 51.8 12 2398.1 430.3 13 >5000 >5000 negative control N / A N / A

[0159] As shown in Table 5, there were significant differences in the expression levels of different recombinant plasmids. SEQ ID Nos. 8, 10, 11, 12, and 13 showed protein expression in both the extracellular supernatant and cell lysis supernatant; while SEQ ID No. 7 showed expression only in the extracellular supernatant, with no protein detected in the cell lysis supernatant. SEQ ID No. 9 showed no protein expression in either the extracellular or lysis supernatant. Of particular note are the very high expression levels of SEQ ID Nos. 10 and 13 in both supernatants, significantly superior to the other groups.

[0160] The difference between SEQ ID No. 7 and SEQ ID No. 9 lies in the use of different signal peptides. SEQ ID No. 7 uses the proinsulin signal peptide, while SEQ ID No. 9 uses the proglucagon signal peptide. Based on the experimental data in Table 6, it can be clearly observed that no protein expression was detected in either the extracellular supernatant or the lysis supernatant of SEQ ID No. 9. Conversely, protein expression was successfully detected in the extracellular supernatant of SEQ ID No. 7. This comparative result indicates that the proinsulin signal peptide plays a positive role in promoting protein expression.

[0161] In recombinant plasmids that successfully expressed proteins in both extracellular supernatant and cell lysis supernatant, SEQ ID No. 8 and SEQ ID No. 13 employed C-terminal extension of Exendin-4, suggesting that C-terminal extension of Exendin-4 may contribute to improving the secretion efficiency of recombinant proteins. Furthermore, both plasmids contained a proinsulin signal peptide, further confirming the positive role of this signal peptide in promoting protein expression.

[0162] In the comparative analysis of short peptide tandem linkages, SEQ ID No. 10 and SEQ ID No. 13 used the G4S linker, while SEQ ID No. 11 and SEQ ID No. 12 used the P2A linker. The results showed that the flexible G4S linker resulted in higher protein expression levels compared to the P2A linker. This finding is significant for optimizing protein expression strategies.

[0163] Overall, these findings indicate that the application of the proinsulin signal peptide in recombinant plasmids can promote the extracellular secretion of proteins. Furthermore, C-terminal extension of Exendin-4 plays a positive role in improving secretion efficiency. Simultaneously, the use of short peptide tandems, particularly the G4S linker, has also shown potential for enhancing secretion efficacy. Therefore, it can be inferred that the strategy of combining the proinsulin signal peptide with C-terminal extension of Exendin-4 and the G4S linker is a superior choice for improving the secretion efficiency of GCG analogs.

[0164] Example 2: mRNA expression

[0165] To advance the development of mRNA drugs, the amino acid sequence shown in SEQ ID No. 13 of Example 1 was selected as the basis for designing the mRNA sequence, as shown in SEQ ID No. 15. This aims to achieve efficient protein expression and maximize the therapeutic effect of the mRNA drug.

[0166] SEQ ID No. 17 and SEQ ID No. 18 were used as control groups. SEQ ID No. 17 was designed based on SEQ ID No. 7 in Example 1, and SEQ ID No. 18 was designed based on SEQ ID No. 8 in Example 1. Sequences are shown in Tables 7 and 8. The mRNA structure in this example was 5'UTR-Kozak-CDS-3'UTR-poly(A).

[0167] Table 7. RNA sequences of major domains in mRNA molecules.

[0168]

[0169] Table 8. RNA sequences expressing GCG

[0170]

[0171]

[0172] (1) Plasmid linearization: Linear mRNA sequences of target genes (mRNAs) 1-3 were synthesized in vitro by a nearshore protein research company. The nucleotide sequences of the CDS regions of linear mRNAs 1-3 are shown in SEQ ID No. 15, SEQ ID No. 17, and SEQ ID No. 18, respectively. SEQ ID No. 15 was designed based on SEQ ID No. 13 in Example 1, SEQ ID No. 17 was designed based on SEQ ID No. 7 in Example 1, and SEQ ID No. 18 was designed based on SEQ ID No. 8 in Example 1. The structure of linear mRNAs 1-3 is 5'UTR-Kozak-CDS-3'UTR-poly(A). The target gene mRNA sequence was inserted into the plasmid to construct the plasmid template. The plasmid template was linearized. The reaction system included 5 μL of 10×buffer, 20 μg of plasmid template, 1 μL of BsaI (20 U / μL), and nuclease-free water to a final volume of 50 μL. The mixture was incubated at 37°C for 30 min, and the linearized plasmid was purified and recovered.

[0173] (2) In vitro transcription: The obtained linearized plasmid was used for in vitro transcription. The reaction system included 2 μL of 10×Transcription Buffer A, 2 μL of adenine triphosphate (ATP) (100 mM), 2 μL of cytosine triphosphate (CTP) (100 mM), 2 μL of guanine triphosphate (GTP) (100 mM), 2 μL of uracil triphosphate (UTP) / modified UTP (100 mM), 2 μL of Enzyme Mix, 1 μg of DNA template, and nuclease-free water to a final volume of 20 μL. The mixture was incubated at 37°C for 2 hours. After the reaction, 2 μL of DNase I (5 U / μL) was added, the mixture was shaken for 3 seconds, and incubated at 37°C for 30 min to digest the transcribed DNA template. The resulting IVT reaction solution was purified to remove impurities such as NTPs, enzymes, proteins, and salt ions before capping. The capping reaction system consisted of 10 μg mRNA, 2 μL 10×Capping Reaction Buffer, S-adenosylmethionine (4 mM), 0.2 μL guanine nucleoside triphosphate (GTP) (100 mM), 1 μL capping enzyme (10 U / μL), 1 μL mRNA 2'-O-methyltransferase (50 U / μL), 0.5 μL RNase inhibitor (40 U / μL), and nuclease-free water to a final volume of 20 μL. The mixture was incubated at 37°C for 1 h.

[0174] RNA concentration was measured using Nanodrop at 1 mg / mL. RNA integrity was assessed by gel electrophoresis. Purity was determined by capillary electrophoresis, with a purity >85%.

[0175] (3) mRNA expression at the cellular level

[0176] Cell culture: 293T cells were cultured in culture dishes using DMEM medium. When the 293T cells grew to 70-90%, the cells were digested with trypsin, dispersed into single cells, and a cell suspension was prepared with a density of 2.5 × 10 cells / mL. 500 μL of cell suspension was added to each well of a 24-well plate and cultured overnight.

[0177] Preparation of transfection premix: First, add 25 μL of Opti-MEM to the centrifuge tube. TM Add 1 μL of lipo3000 transfection reagent dropwise to the culture medium, mix well by pipetting, and prepare a liposome premix. Add 25 μL of Opti-MEM to the centrifuge tube. TM In the culture medium, slowly add 2 μg mRNA and 1 μL of transfection reagent Lipo 3000, mix well by pipetting, and prepare an mRNA premix. Add the liposome premix to the mRNA premix, mix well by pipetting, and prepare an mRNA-liposome complex (50 μL total). Mix well by pipetting and let stand for 10 min.

[0178] Transfection loading: Slowly add 50 μL of the mRNA-liposome complex to the cell suspension, mix gently, and incubate at 37°C for 24 h, 72 h, or 196 h.

[0179] The detection was performed using the Glucagon ELISA kit (Lianke Biotechnology): Standard reconstitution: Reconstitute with purified water to 10000 pg / mL according to the label on the vial; Standard dilution: Serially dilute the standards using cell culture medium, performing 2-fold dilutions in 8 gradients (5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312.5 pg / mL, 156.25 pg / mL, 78.13 pg / mL, 0 pg / mL); Standard loading: Add 100 μL / well to the ELISA plate; Sample loading: ① Extracellular supernatant: After centrifugation, add 100 μL / well to the ELISA plate; ② Intracellular lysis supernatant: Add 120 μL of lysis buffer, lyse for 5 min, centrifuge, and add 100 μL / well to the ELISA plate. Cover the plate with a sealing film and incubate at 25℃ and 200rpm for 2 hours. Add detection antibody: Dilute the detection antibody 1:100 with 1× detection buffer and add 100μL / well to the microplate. Cover the plate with a sealing film and incubate at 25℃ and 200rpm for 1 hour. Add streptavidin: Dilute streptavidin 1:100 with 1× detection buffer and add 100μL / well to the microplate. Cover the plate with a sealing film and incubate at 25℃ and 200rpm for 45 minutes. For color development: Add 100μL / well SubstrateTMB and incubate at room temperature in the dark for about 10 minutes. Add 100μL / well StopSolution to stop the incubation and immediately place the plate in a microplate reader to read the OD value.

[0180] Table 9 shows the OD values ​​detected after mRNA transfection into cells and the calculated protein concentrations.

[0181] Table 9. GCG protein expression levels at different time points after mRNA transfection into cells.

[0182]

[0183] Table 9 presents a comparative analysis of different mRNA expression levels. Linear mRNA-2 and mRNA-3 were used as control groups. The CDS sequence of mRNA-2 (SEQ ID No. 17) was designed based on SEQ ID No. 7 in Example 1, and the CDS sequence of mRNA-3 (SEQ ID No. 18) was designed based on SEQ ID No. 8 in Example 1. Compared to SEQ ID No. 17 and SEQ ID No. 18, linear mRNA-1 with the CDS sequence shown in SEQ ID No. 15 consistently exhibited higher protein expression levels in both the extracellular supernatant and lysis supernatant from 24 to 196 hours post-transfection. This result indicates that the GCG-G4S linker-GCG-Exendin-4C-terminal extension design can effectively improve mRNA expression efficiency.

[0184] Example 3 Synthesis of Circular RNA

[0185] I. Sequence Design of Linear Precursor RNA

[0186] To advance the development of circular RNA drugs, the amino acid sequence shown in SEQ ID No. 13 of Example 1 was selected as the basis for designing circular RNA sequences. Circular RNAs were designed to achieve efficient protein expression and maximize the therapeutic efficacy of circular RNA drugs.

[0187] Specifically, the linear mRNA-1 sequence in Example 2 was modified into a circular RNA, and the mRNA-2 and mRNA-3 in Example 2 were modified into circular RNAs as control groups, and named circular RNA-1, circular RNA-2 and circular RNA-3, respectively. Circular RNA-1, circular RNA-2 and circular RNA-3 have the same CDS sequence as linear mRNA-1, linear mRNA-2 and linear mRNA-3, respectively.

[0188] The linear precursor RNA construct used for circularization comprises, from the 5' end to the 3' end: a 5' outer homologous arm, a 3' intron, an E2 exon, a 5' spacer region, a Kozak, an internal ribosome entry site (IRES), a coding region (CDS), a 3' spacer region, an E1 exon, a 5' intron, and a 3' outer homologous arm; wherein the coding region encodes the GCG fusion protein. The coding region sequence is shown in Table 8.

[0189] The structure of the circular RNA after circularization is as follows: exon E2, 5' spacer, internal ribosome entry site (IRES), Kozak, coding region (CDS), 3' spacer, and exon E1 connected in sequence; wherein the coding region encodes the GCG fusion protein. The coding region sequence is shown in Table 8.

[0190] Table 10. RNA sequences of circular elements in circular RNA molecules.

[0191]

[0192]

[0193] II. Preparation of linear precursor RNA

[0194] (1) Construction of circular plasmids: Circular RNA-1, Circular RNA-2, and Circular RNA-3 of the target genes were synthesized in vitro by a nearshore protein research company. The CDS sequence of Circular RNA-1 was designed based on SEQ ID No. 13 in Example 1, and is shown in SEQ ID No. 15. The CDS sequence of Circular RNA-2 was designed based on SEQ ID No. 7 in Example 1, and is shown in SEQ ID No. 17. The CDS sequence of Circular RNA-3 was designed based on SEQ ID No. 8 in Example 1, and is shown in SEQ ID No. 18. All three structures are CVB3-CDS. The circular plasmids were based on the pCDNA3.1 backbone, and the PIE circularization system derived from the Cyanobacterium type I intron sequence was inserted into Hind III and Apa I to form circular plasmids.

[0195] (2) Plasmid linearization: The reaction system included 5 μL of 10× buffer, 20 μg of plasmid template, 1 μL of BsaI (20 U / μL), and nuclease-free water to a final volume of 50 μL. The mixture was incubated at 37 °C for 30 min, and the linearized plasmid was purified and recovered.

[0196] The supercoiled plasmid was cut with restriction endonucleases to obtain a linearized template. The reaction system is shown in Table 11. The reaction conditions were incubation at 37℃ for 30 min. The linearized plasmid was then recovered.

[0197] Table 11

[0198]

[0199]

[0200] (3) In vitro transcription: Using the linearized plasmid product obtained in step (2) as an in vitro transcription template, in vitro transcription was performed using the T7high yield RNA synthesis kit to obtain linear RNA molecules. The reaction system is shown in Table 12. After mixing, the reaction was carried out in a PCR instrument under the following conditions:

[0201] Table 12

[0202] reaction system volume ATP 2μL CTP 2μL GTP 2μL UTP 2μL pDNA / PCR template 1μg 10×transcription Buffer 2μL T7 Enzyme Mix 2μL RNase-free water Up to 20 μL

[0203] Table 13

[0204] Reaction conditions time 37℃ 3 hours 4℃ ∞

[0205] (4) DNase I digestion

[0206] Remove template DNA by treating with DNase I for 15 min. Reaction conditions: Add 1 μL of DNase I (1 U / μL) to the IVT product, mix well, and react at 37°C for 15 min.

[0207] (5) RNA precipitation with LiCl. Add 40 μL of DEPC water to the DNase I digestion system and transfer to a 1.5 mL RNase-free EP tube. Add 30 μL of 7.5 M LiCl solution, mix well, and incubate at -20°C for 30 min. Centrifuge at 12,000 rpm for 10 min at 4°C and discard the supernatant. Wash the precipitate once with 1 mL of 80% ethanol and centrifuge at 12,000 rpm for 5 min at 4°C. Discard the supernatant, briefly centrifuge for 10 seconds in a handheld centrifuge, and remove the remaining liquid with a pipette. Air-dry the RNA at room temperature for 5 min, add 150 μL of DEPC water, and incubate on ice for 20 min to dissolve the RNA. Measure the RNA concentration using Nanodrop.

[0208] III. Preparation of Circular RNA

[0209] Prepare a 10× Circularization buffer: 500 mM Tris-HCl, 100 mM MgCl2, 10 mM DTT, pH 7.5. Take 50 μg of the RNA synthesized in vitro in step (4) for in vitro circularization reaction. The reaction system is as follows:

[0210] Table 14

[0211] reaction system volume 10×Circularization buffer 5μL IVT RNA 50μg DEPC water up to 50μL

[0212] Mix the above system thoroughly and incubate at 50°C for 10 min in a PCR instrument. Use the LiCl precipitation method from step (4) to recover RNA, adding 30 μL of DEPC water to dissolve the RNA. Measure the RNA concentration using Nanodrop; the concentration is 1 mg / mL. Gel electrophoresis is used to assess RNA integrity. Capillary electrophoresis is used to determine purity; the purity is >80%.

[0213] IV. Expression of Circular RNA at the Cellular Level

[0214] Cell culture: 293T cells were cultured in culture dishes using DMEM medium. When the 293T cells grew to 70-90%, the cells were digested with trypsin, dispersed into single cells, and a cell suspension was prepared with a density of 2.5 × 10 cells / mL. 500 μL of cell suspension was added to each well of a 24-well plate and cultured overnight.

[0215] Preparation of transfection premix: First, add 25 μL of Opti-MEM to the centrifuge tube. TM Add 1 μL of lipo3000 transfection reagent dropwise to the culture medium, mix well by pipetting, and prepare a liposome premix. Add 25 μL of Opti-MEM to the centrifuge tube. TM In the culture medium, slowly add 2 μg of circular RNA and 1 μL of transfection reagent Lipo 3000, mix well by pipetting, and prepare an mRNA premix. Add the liposome premix to the circular RNA premix, mix well by pipetting, and prepare a circular RNA-liposome complex, totaling 50 μL. Mix well by pipetting and let stand for 10 min.

[0216] Transfection loading: Slowly add 50 μL of the circular RNA-liposome complex to the cell suspension, mix gently, and incubate at 37°C for 24 h, 72 h, or 196 h.

[0217] The detection was performed using the Glucagon ELISA kit (Lianke Biotechnology): Standard reconstitution: Reconstitute with purified water to 10000 pg / mL according to the label on the vial; Standard dilution: Serially dilute the standards using cell culture medium, performing 2-fold dilutions in 8 gradients (5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312.5 pg / mL, 156.25 pg / mL, 78.13 pg / mL, 0 pg / mL); Standard loading: Add 100 μL / well to the ELISA plate; Sample loading: ① Extracellular supernatant: After centrifugation, add 100 μL / well to the ELISA plate; ② Intracellular lysis supernatant: Add 120 μL of lysis buffer, lyse for 5 min, centrifuge, and add 100 μL / well to the ELISA plate. Cover the plate with a sealing film and incubate at 25℃ and 200rpm for 2 hours. Add detection antibody: Dilute the detection antibody 1:100 with 1× detection buffer and add 100μL / well to the microplate. Cover the plate with a sealing film and incubate at 25℃ and 200rpm for 1 hour. Add streptavidin: Dilute streptavidin 1:100 with 1× detection buffer and add 100μL / well to the microplate. Cover the plate with a sealing film and incubate at 25℃ and 200rpm for 45 minutes. For color development: Add 100μL / well SubstrateTMB and incubate at room temperature in the dark for about 10 minutes. Add 100μL / well Stop Solution to stop the incubation and immediately read the OD value using a microplate reader. Table 15 shows the OD values ​​detected after mRNA transfection and the protein concentration calculated based on the OD values.

[0218] Table 15. GCG protein expression levels at different time points after circRNA transfection.

[0219]

[0220] As shown in Table 15, circular RNA-1 maintained high protein expression levels for up to 196 hours post-transfection, particularly during the first 24 to 196 hours. GCG expression levels in both the extracellular and lysis supernatants significantly exceeded those of circular RNA-2 and circular RNA-3. The higher expression levels in the extracellular supernatant compared to the lysis supernatant indicate that the proinsulin signal peptide plays a positive role in promoting protein expression. This finding strongly demonstrates that the GCG-G4S linker-GCG-Exendin-4C-terminal extension design significantly enhances the expression efficiency of circular RNA. Furthermore, compared to mRNA, the circular RNA structure is more stable, effectively prolonging the half-life of the GCG fusion protein.

[0221] Example 4 Functional Activity Detection

[0222] The functional activity of the GCG fusion protein was determined in the HEK-293 clone cell line. The Ultra cAMP kit (PerkinElmer) measures the accumulation of cAMP.

[0223] Cell culture: 293T cells were cultured in culture dishes using DMEM medium. When the 293T cells grew to 70-90%, the cells were digested with trypsin, dispersed into single cells, and a cell suspension was prepared with a density of 2.5 × 10 cells / mL. 500 μL of the cell suspension was added to a 24-well plate and incubated overnight.

[0224] Preparation of transfection premix: First, add 25 μL of Opti-MEM medium to a centrifuge tube, then slowly add 1.5 μL of RNAMAX transfection reagent, and mix thoroughly by pipetting to prepare the liposome premix. Add 25 μL of Opti-MEM to the centrifuge tube. TM In the culture medium, slowly add 2 μg of linear mRNA-1 and circular RNA-1 dropwise, mix well by pipetting, and prepare an RNA premix. Add liposome premix to the RNA premix, mix well by pipetting, and prepare a DNA-liposome complex, totaling 50 μL. Mix well by pipetting and let stand for 10-20 min.

[0225] Transfection: 50 μL of the RNA-liposome complex was slowly added to the cell suspension, gently mixed, and incubated at 37°C. Cell supernatants were collected after 24 h, 48 h, 72 h, and 120 h. The cell supernatants were centrifuged at 1000 rpm for 5 min, cell debris was discarded, and the extracellular supernatant was obtained.

[0226] cAMP assay of GCG fusion protein in cell supernatant: Cells overexpressing GCGR were resuspended in activation solution at a density of 6 × 10⁻⁶. 5 / mL. Cells were aliquoted into 384 Optiplates (PerkinElmer) (3000 cells per well). 5 μL of cell supernatant transfected with RNA expressing the GCG fusion protein was added to each well, with replicates for each sample, and incubated at room temperature for 40 min. Subsequently, 5 μL of ULight-cAMP tracer and 5 μL of ULight-anti-cAMP were added, and incubation continued at room temperature for 1 h. Signal was detected using an EnVision multi-label plate reader (PerkinElmer). The amount of cAMP generated in each well (nM) was converted to the percentage of the maximum response observed with human GCG. The percentage of maximum response was compared with the concentration of the added cell supernatant (2 μg RNA transfected cells were used as the initial concentration as 1M, followed by 5-fold dilutions for a total of 8 gradients). Data were transformed using a cAMP standard curve and analyzed using a three-parameter logistic equation. The results are shown in Table 14.

[0227] Table 14. Agonistaltic activity of RNA-expressed GCG fusion proteins on GCG receptors

[0228]

[0229] Linear mRNA-1 and circular RNA-1 encode the same amino acid sequence (i.e., SEQ ID No. 13). Because the former is mRNA and the latter is circular RNA, their base numbers and molecular weights differ. In this embodiment, the activation levels of the fusion proteins expressed by these two RNAs for GCGR were measured at different time points. The results are shown in Table 14. Linear mRNA-1 reached its lowest EC50 value (0.02030) 24 hours after transfection and then slightly increased over time; while circular RNA-1 unexpectedly showed the strongest activity at an EC50 value of 0.1048 48 hours after transfection, which may be related to the unique properties of circular RNA.

[0230] It can be seen that both linear mRNA-1 and circular RNA-1 can successfully express the target protein and effectively activate the receptor. This further demonstrates that using the G4S linker, C-terminal extension, and repetitive sequences, both mRNA and circRNA can express the target protein, and the fusion proteins expressed by mRNA and circRNA have comparable activity in activating the receptor.

[0231] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A glucagon fusion protein, characterized in that, Includes the following structure: Formula (I): [GCG]-[(L)-(GCG)] m -[X] n ; GCG stands for glucagon fragment, and L stands for linker peptide; X is the amino acid sequence shown in SEQ ID No. 2; m is an integer ≥ 0, n is 0 or 1, and m and n are not both 0.

2. The glucagon fusion protein according to claim 1, characterized in that, The amino acid sequence of each GCG is independently shown in SEQ ID No. 1; Optionally, the linker peptide is a flexible linker peptide; Optionally, the linker peptide is (GGGGS). x x is a positive integer ≥ 1; Optionally, the glucagon fusion protein includes the following structure: Formula (i): [GCG]-[X]; or, Equation (ii): [GCG]-[(L)-(GCG)]; or, Formula (ⅲ): [GCG]-[(L)-(GCG)]-[X]; Optionally, the amino acid sequence of the glucagon fusion protein includes the sequence shown in SEQ ID No. 39, 40 or 41.

3. A polynucleotide, characterized in that, CDS containing coded (II) or (III) structures: Formula (II): [Y]-[GCG]-[(L1)-(GCG)] m -[X] n ;or, Formula (III): [Y]-[GCG]-[(L2)-(Y)-(GCG)] m -[X] n ; Y is the signal peptide, GCG is the glucagon fragment, L1 is the flexible linker peptide, L2 is the cleavable linker peptide, and X is the amino acid sequence shown in SEQ ID No.

2. m is an integer ≥ 0, n is 0 or 1, and m and n are not both 0; Optionally, n is 0 in equation (Ⅲ); Optionally, the linker peptide L1 is (GGGGS). x x is a positive integer ≥ 1; Optionally, signal peptide Y is a pre-proinsulin signal peptide; Optionally, the amino acid sequence of the proinsulin signal peptide is shown in SEQ ID No.

3.

4. The polynucleotide according to claim 3, characterized in that, The polynucleotides include DNA; Optionally, the DNA sequence encoding GCG is shown in SEQ ID NO. 24 or 25; Optionally, the DNA sequence encoding the proinsulin signal peptide is shown in SEQ ID NO.26; Optionally, the DNA sequence encoding the linker peptide L1 is shown in SEQ ID NO.27; Optionally, the DNA sequence encoding X is shown in SEQ ID NO.28; Optionally, the polynucleotide is DNA, and the nucleotide sequence is as shown in SEQ ID NO. 21 or 23.

5. The polynucleotide according to claim 3, characterized in that, The polynucleotides include RNA; Optionally, the RNA sequence encoding GCG is shown in SEQ ID NO.29 or 30; Optionally, the RNA sequence encoding the proinsulin signal peptide is shown in SEQ ID NO.31; Optionally, the RNA sequence encoding the linker peptide L1 is shown in SEQ ID NO.32; Optionally, the RNA sequence encoding X is shown in SEQ ID NO.33; Optionally, the RNA includes mRNA or circular RNA; Optionally, the nucleotide sequences of the CDS of the mRNA or circular RNA are independently shown as SEQ ID NO. 15 or 18, respectively; Optionally, the structure of the mRNA includes: 5'UTR-Kozak-CDS-3'UTR-polyA; Optionally, the nucleotide sequence of the 5'UTR is shown in SEQ ID NO.34; Optionally, the nucleotide sequence of Kozak is GCCACC; Optionally, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.36; Optionally, the nucleotide sequence of polyA is shown in SEQ ID NO.35; Optionally, the structure of the circular RNA includes: E2 exon-5' spacer region-internal ribosome entry site-Kozak-CDS-3' spacer region-E1 exon; Optionally, the nucleotide sequence of exon E2 is shown in SEQ ID NO.19; Optionally, the nucleotide sequence of the 5' spacer region is shown in SEQ ID NO.37; Optionally, the nucleotide sequence of the internal ribosome entry site is shown in SEQ ID NO.14; Optionally, the nucleotide sequence of the 3' spacer region is shown in SEQ ID NO.38; Optionally, the nucleotide sequence of exon E1 is shown in SEQ ID NO.

20.

6. A carrier, characterized in that, The vector carries the polynucleotide as described in any one of claims 3 to 5.

7. A cell, characterized in that, The cell expresses the glucagon fusion protein of claim 1 or 2, or carries the polynucleotide of any one of claims 3 to 5, or contains the vector of claim 6.

8. A method for preparing a glucagon fusion protein, characterized in that, This includes expressing the glucagon fusion protein in cells; Optionally, the preparation method includes introducing the polynucleotide according to any one of claims 3 to 5 into cells; Optionally, the polynucleotide is mRNA, and the preparation method further includes in vitro transcription to prepare mRNA; Optionally, the polynucleotide is a circular RNA, and the preparation method further includes forming a circular RNA through type I introns.

9. The use of the glucagon fusion protein of claim 1 or 2, or the polynucleotide of any one of claims 3 to 5, or the carrier of claim 6, or the cell of claim 7, or the preparation method of claim 8 in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition is used for the prevention and / or treatment of hypoglycemia. The pharmaceutical composition is used for the prevention and / or treatment of metabolic disorders; Optionally, the metabolic disorder includes hypoglycemia, type 1 diabetes, kidney disease caused by metabolic disorder, fatty liver, or obesity.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the glucagon fusion protein according to claim 1 or 2, or the polynucleotide according to any one of claims 3 to 5, or the carrier according to claim 6, or the cell according to claim 7; the pharmaceutical composition is used for the prevention and / or treatment of metabolic disorders. Optionally, the metabolic disorder includes hypoglycemia, type 1 diabetes, kidney disease caused by metabolic disorder, fatty liver, or obesity; Optionally, the polynucleotide includes mRNA or circular RNA; Optionally, the nucleotide sequences of the CDS of the mRNA or circular RNA are independently shown as SEQ ID NO. 15 or 18, respectively; Optionally, the pharmaceutical composition further includes a GLP-1 receptor agonist and / or a GIP receptor agonist.

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