Cyclic rnas encoding glp-1 and uses thereof

Abstract

This application provides a circular RNA encoding GLP-1 and its use in treating diseases such as obesity, diabetes, and fatty liver disease or steatohepatitis associated with metabolic dysfunction.

Description

Technical Field

[0001] This application relates to a circular RNA encoding GLP-1 and its application in the treatment of diseases. Background Technology

[0002] Glucagon-like peptide-1 (GLP-1) is a 31-amino acid-long incretin secreted by intestinal L cells and pancreatic α cells. GLP-1 induces insulin release to regulate blood glucose and inhibits glucagon secretion by binding to GLP-1 receptors (GLP1Rs) widely expressed in the pancreas, central nervous system, stomach, and muscles, thereby delaying gastric emptying and reducing food consumption (Müller, TD et al., Molecular metabolis. 2019). Therefore, GLP-1 receptor agonists (GLP-1RAs) can reduce body weight and blood glucose levels, thus holding promise as therapeutic agents for type 2 diabetes and obesity. Furthermore, they have shown potential in numerous clinical trials for treating metabolic dysfunction-associated fatty liver disease (MAFLD) or steatohepatitis (MASH). However, due to its susceptibility to N-terminal degradation by the endogenous enzyme dipeptidyl peptidase-4 (DPP-4), the natural GLP-1 peptide has a short half-life in plasma, approximately 1-2 minutes (Lee, Seungah, and Dong Yun Lee. Annals of pediatric endocrinology & metabolism. 2017). Therefore, truncated GLP-1 loses its affinity for GLP1R and cannot maintain its role in glucose homeostasis. Liraglutide and semaglutide are long-acting GLP-1RAs that achieve extended half-lives (approximately 14 hours and 160 hours, respectively) by preventing degradation through site-specific PEGylation and site mutation (Nauck, Michael A et al., Molecular metabolism. 2021).

[0003] Despite its therapeutic successes, there is a general concern in clinical practice that semaglutide may lead to a reduction in lean muscle mass along with weight loss, which can account for 5-50% of total body weight (Sargeant, Jack Alistair et al., Endocrinology and metabolism (Seoul, Korea), 2019; McCrimmon, Rory J et al., Diabetologia, 2020; Wilding, John PH et al., Journal of the Endocrine Society, 2021). Lean muscle primarily plays a role in maintaining body support, especially in older adults, and is also involved in glucose metabolism. Furthermore, better weight and glycemic control have been observed in patients with well-adhered type 2 diabetes (T2D). Multiple studies have reported that patients with type 2 diabetes (T2D) treated with GLP-1RAs once a week have better adherence than those treated twice or once a day (Alatorre, Carlos et al., Diabetes, obesity & metabolism, 2017; Nguyen, Hiep et al., Advances in therapy, 2017). However, after 360 days of treatment, adherence to duraglutide or smegglutide dropped to only 40% (Uzoigwe, Chioma et al., Diabetes therapy: research, treatment and education of diabetes and related disorders, 2021). Extending the dosing interval is associated with improved patient adherence. Therefore, there is a need for a new drug with a lower dosing frequency that can treat type 2 diabetes and obesity while maintaining lean muscle mass. Summary of the Invention

[0004] This application relates to a novel circular RNA encoding GLP-1 or a variant thereof, which possesses improved pharmaceutical properties compared to recombinant proteins and / or peptides. The circular RNA of this application exhibits superior efficacy compared to the approved commercial product semaglutide and addresses the issue of semaglutide reducing lean muscle mass. The circular RNA of this application has the effect of reducing body weight without reducing lean muscle mass, and reducing body fat without reducing lean muscle mass.

[0005] In one aspect, this application provides a circular RNA comprising a regulatory element and an expression element, wherein the expression element comprises a polynucleotide encoding GLP-1 or a variant thereof.

[0006] In one aspect, this application provides a composition comprising the circular RNA disclosed herein, wherein the composition further comprises a pharmaceutically acceptable excipient.

[0007] In one aspect, this application provides a method for treating a subject's disease, comprising administering to the subject a therapeutically effective amount of the circular RNA or composition disclosed in this application.

[0008] In one aspect, this application provides the use of the circular RNA or composition disclosed herein in the preparation of a medicament for treating a subject’s disease.

[0009] In one aspect, this application provides the circular RNA or composition disclosed herein for use in treating a subject's disease.

[0010] In one aspect, this application provides a method for reducing the weight of a subject without reducing the subject's lean muscle mass, the method comprising administering to the subject a therapeutically effective amount of the circular RNA or composition disclosed in this application.

[0011] In one aspect, this application provides a method for reducing a subject's fat without reducing the subject's lean muscle mass, the method comprising administering to the subject a therapeutically effective amount of the circular RNA or composition disclosed in this application. Attached Figure Description

[0012] Figure 1 This is an overview diagram of plasmids used to produce circular RNA.

[0013] Figure 2 To analyze the circular RNA-GLP products after ribonuclease R treatment by urea-polyacrylamide gel electrophoresis.

[0014] Figure 3 The expression of GLP-1 products in the supernatant of 293T cells transfected with the corresponding circular RNA-GLP is shown.

[0015] Figure 4 The circular RNA-GLP003-encoded GLP-1 product can induce ERK phosphorylation in 293T(A) and 3T3-L1(B) cells transfected with the human GLP-1 receptor.

[0016] Figure 5 The circular RNA-GLP003-encoded GLP-1 product can bind to the human GLP-1 receptor, activating downstream signaling pathways and thereby increasing luciferase activity.

[0017] Figure 6 The study showed that circular RNA-GLP003 reduced weight gain in Western diet-induced BKS-db / db mice.

[0018] Figure 7 The results showed that on day 14 after administration, circular RNA-GLP003 reduced serum total cholesterol levels in Western diet-induced BKS-db / db mice.

[0019] Figure 8 The study showed that circular RNA-GLP003 reduced the body weight of DIO mice.

[0020] Figure 9 The results showed that circular RNA-GLP003 reduced fat mass in DIO mice (A) without altering lean meat mass (B).

[0021] Figure 10 The study showed that circular RNA-GLP003 inhibited food intake in DIO mice.

[0022] Figure 11 The study showed that circular RNA-GLP003 reduced fasting blood glucose levels in DIO mice.

[0023] Figure 12 (A and B) show that circular RNA-GLP003 increases c-fos activity in mouse brain regions. Detailed Implementation

[0024] Unless otherwise defined below, all technical and scientific terms used in this application have the meanings commonly understood by those skilled in the art. The term "technique" as used herein is intended to refer to techniques generally understood in the art, including modifications or equivalent substitutions that are obvious to those skilled in the art. While those skilled in the art should be able to readily understand the following terms, the definitions are provided below for better explanation of this application.

[0025] As used in this application, the terms “comprising,” “including,” “having,” “containing,” or “including,” and variations thereof, are inclusive or open-ended and do not exclude other elements or method steps not listed.

[0026] As used herein, the terms “implementation,” “disclosed in this application,” or “disclosure” are not intended to be limiting but are generally applicable to any embodiment as defined in the claims or described in this application. These terms may be used interchangeably in this application.

[0027] As used in this application, the terms "treat / treating / treatment" refer to the elimination, reduction, or improvement of a disease or condition, and / or related symptoms. While not absolutely prohibited, treating a disease or condition does not require the elimination of that disease, condition, or related symptoms. The term "treatment" and its synonyms refer to the administration of a therapeutically effective amount of the circular RNA or composition disclosed in this application to a subject who needs to receive the treatment. Treatment can be directed at symptoms, for example, suppressing symptoms. Symptoms can be affected in the short term, treated in the medium term, or treated long-term, such as in a maintenance therapy setting.

[0028] Throughout the specification, the term "a / an" refers to one or more of the same entity; for example, "(a) nucleotide" is understood to mean one or more polynucleotides. Therefore, the terms "a / an," "one or more," and "at least one" are used interchangeably in this application.

[0029] As used herein, the term "variant" refers to a peptide that differs from the described peptide due to amino acid substitutions, deletions, insertions, and / or modifications. Variants can be generated using mutagenesis techniques known in the art.

[0030] The term "composition" or "pharmaceutical composition" refers to a composition comprising the circular RNA provided in this application, together with, for example, a pharmaceutically acceptable carrier, excipient, or diluent, for administration to a subject in need of treatment.

[0031] The term "pharmaceutically acceptable" means that, within reasonable medical judgment, exposure to human and animal tissues is appropriate without causing excessive toxicity or other complications commensurate with a reasonable benefit / risk ratio.

[0032] "Effective dose" refers to the amount of the circular RNA provided in this application, which, when administered to a subject as a single dose or a portion of a series of doses, is effective in treating the condition. For example, in the case of obesity, the dose of the drug is effective if taking the drug results in weight loss or maintenance of weight (e.g., prevention of weight gain), reduction of body fat, prevention or regulation of hypoglycemia, prevention or regulation of hyperglycemia, promotion of insulin synthesis, or reduction of food intake, or if so, the dose may be a fixed dose for all subjects to be treated; or it may vary depending on the subject's weight, health status and physical condition, desired weight loss or weight maintenance, the formulation of the circular RNA or composition disclosed in this application, a professional assessment of the medical condition, and other relevant factors.

[0033] The term "subject" refers to any subject who requires treatment with the circular RNA or composition provided in this application, particularly a mammalian subject. Mammal subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cattle, apes, monkeys, orangutans, chimpanzees, etc. In one embodiment, the subject is a human subject.

[0034] The term "YTE mutation" refers to a mutation at the CH2-CH3 interface that forms a contact residue for FcRn binding. According to the EU index of Kabat numbers, the YTE mutation for IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc is M252Y / S254T / T256E.

[0035] The term "circular RNA" can refer to both modified and unmodified circular RNA. RNA modification is known in the art, and those skilled in the art can choose appropriate modifications if necessary.

[0036] As used in this application, the term “about” includes ±10% of the stated number. Therefore, “about 10” refers to 9-11.

[0037] The terms “metabolic dysfunction-associated steatohepatitis (MASH)” and “non-alcoholic steatohepatitis (NASH)” are used interchangeably. Those skilled in the art will know that MASH is also known as NASH.

[0038] The terms “metabolic dysfunction-associated fatty liver disease (MAFLD)” and “non-alcoholic fatty liver disease (NAFLD)” are used interchangeably. Those skilled in the art will know that MAFLD is also known as NAFLD.

[0039] The term "lean muscle mass" refers to the amount of muscle that makes up body composition. It can be used interchangeably with the term "lean body mass".

[0040] Circular RNA As used herein, the terms "circRNA," "circular polynucleotide," or "circular RNA" are used interchangeably and refer to polynucleotides that form a circular structure through covalent or non-covalent bonds. When circular RNA is mentioned, those skilled in the art will understand that the polynucleotide in RNA refers to a polynucleotide.

[0041] In one aspect, this application provides a circular RNA comprising a regulatory element and an expression element, said expression element comprising a polynucleotide encoding GLP-1 or a variant thereof.

[0042] In some embodiments, the circular RNA comprises a nucleotide sequence as shown in SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0043] In some embodiments, the polynucleotide encoding GLP-1 or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 22, 23, 24, 25, 26, 27, or 28, preferably the polynucleotide encoding GLP-1 or a variant thereof as shown in SEQ ID NO: 22, 23, 24, 25, 26, 27, or 28.

[0044] In some embodiments, the amino acid sequence of GLP-1 or a variant thereof is shown in SEQ ID NO: 21.

[0045] In some embodiments, the expression element comprises a polynucleotide encoding Fc. In some embodiments, Fc contains one or more mutations. In some embodiments, Fc contains a YTE mutation. In some embodiments, Fc is selected from IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc. In some preferred embodiments, Fc is IgG1 Fc or IgG4 Fc. In some embodiments, the amino acid sequence of Fc is as shown in SEQ ID NO: 30, 31, 32, or 33.

[0046] In some embodiments, the expression element comprises a polynucleotide encoding a linker between GLP-1 or a variant thereof and the Fc fragment. In some embodiments, the linker is GGGGSGGGGSGGGGS. In some embodiments, the circular RNA comprises, in the following order (from 5' to 3'): a polynucleotide encoding GLP-1 or a variant thereof, a polynucleotide encoding the linker, and a polynucleotide encoding the Fc fragment.

[0047] The circular RNA disclosed in this application can be prepared using methods in the prior art. In a preferred embodiment, the circular RNA is prepared using the method described in the embodiments of this application.

[0048] Compared with the control group, the circular RNA disclosed in this application has the beneficial effects of reducing body weight without reducing lean muscle mass and / or reducing fat without reducing lean muscle mass.

[0049] The circular RNA disclosed in this application has the effects of reducing weight, lowering total cholesterol levels, reducing fasting blood glucose concentration, inhibiting food intake, and / or reducing fat without changing lean muscle mass.

[0050] In some embodiments, the circular RNA disclosed in this application comprises two sequences: the sequence shown in SEQ ID NO: 6; and another sequence selected from any of the sequences shown in SEQ ID NOs: 7-20 and 22-28. In a preferred embodiment, the circular RNA disclosed in this application comprises the sequence shown in SEQ ID NO: 6 and the sequence shown in SEQ ID NO: 9.

[0051] Control element A regulatory element may include a sequence located adjacent to an expression element that encodes an expressed product. The regulatory element may be operatively linked to a neighboring sequence. The regulatory element may increase the amount of product expressed compared to the amount expressed in the absence of a regulatory element. Furthermore, a single regulatory element may increase the amount of product expressed by multiple tandem expression sequences. Therefore, a single regulatory element may enhance the expression of one or more expression sequences.

[0052] In some embodiments, the regulatory element is an internal ribosome entry site (IRES) or a fragment thereof.

[0053] A suitable IRES element contained in a cyclic polynucleotide comprises an RNA sequence capable of binding to a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

[0054] In some embodiments, the IRES is selected from: Coxsackievirus B3 (CVB3) IRES, Enterovirus 71 (EV71) IRES, Encephalomyocarditis Virus (EMCV) IRES, Picornavir Virus (PV) IRES, Hepatitis C Virus (HCV) IRES, Adenovirus (AdV) IRES, Human Papillomavirus 31 (HPV31) IRES, Human Herpesvirus (HHV) IRES, Raul's Sarcoma Virus (RSV) IRES, Classical Swine Fever Virus (CSFV) IRES, FGF9 IRES, SLC7A1 IRES, and RUNX1 IRES. In a preferred embodiment, the IRES is a CVB3 IRES (SEQ ID NO: 6).

[0055] The signal peptide can guide the GLP-1 or its variants disclosed in this application into the secretory pathway of host cells. The polynucleotide encoding the signal peptide is linked to the RNA sequence encoding GLP-1 or its variants disclosed in this application in the correct reading frame. The polynucleotide encoding the signal peptide is typically located at the 5' end of the RNA sequence encoding GLP-1 or its variants disclosed in this application.

[0056] In some embodiments, the circular RNA comprises, from 5' to 3', a polynucleotide encoding a signal peptide and a polynucleotide encoding GLP-1 or a variant thereof.

[0057] In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 29.

[0058] Administering circular RNA to subjects In one aspect, this application provides a composition comprising the circular RNA as described in any of the preceding claims, wherein the composition further comprises a pharmaceutically acceptable excipient.

[0059] In some embodiments, the composition comprises nanoparticles, such as lipid nanoparticles.

[0060] In some embodiments, the circular RNA is administered either as naked circular RNA or as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. In non-limiting examples, the pharmaceutically acceptable excipient is polyethyleneimine (PEI) or lipid nanoparticles (LNP). Other examples of liposomes that can be used to administer the circular RNA or composition include: protamine, cationic nanoemulsions, modified dendritic macromolecular nanoparticles, protamine liposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid nanoparticles, cationic lipid-cholesterol nanoparticles, cationic lipid-cholesterol PEG nanoparticles, cationic lipid transfection reagents sold under the trademark LIPOFECTAMINE, non-liposome transfection reagents sold under the trademark FUGENE, or any combination thereof, all of which can be used as pharmaceutically acceptable excipients.

[0061] In some embodiments, the circular RNA or composition disclosed in this application may be administered systemically, locally, superficially, intravenously, intramuscularly, subcutaneously, or by inhalation.

[0062] In some embodiments, the pharmaceutical composition may optionally contain one or more other active substances, such as substances with therapeutic and / or preventative activities.

[0063] In some embodiments, the pharmaceutical composition comprises 1 μg to 500 mg of the circular RNA disclosed in this application, for example, 1 μg, 2 μg, 5 μg, 10 μg, 20 μg, 50 μg, 100 μg, 200 μg, 500 μg, 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or an amount of circular RNA between any of the above amounts.

[0064] In some embodiments, the circular RNA or composition disclosed in this application may be administered in amounts from about 0.0005 mg / day to about 5000 mg / day, for example, about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 mg / day, or any amount between these amounts.

[0065] In some embodiments, the circular RNA or composition disclosed in this application may be administered at a dose of about 1 ng / kg to about 200 mg / kg, about 1 μg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg, for example, at a dose of about 1 μg / kg, about 10 μg / kg, about 25 μg / kg, about 50 μg / kg, about 75 μg / kg, about 100 μg / kg, about 125 μg / kg, about 150 μg / kg, about 175 μg / kg, about 2 00 μg / kg, approximately 225 μg / kg, approximately 250 μg / kg, approximately 275 μg / kg, approximately 300 μg / kg, approximately 325 μg / kg, approximately 350 μg / kg, approximately 375 μg / kg, approximately 400 μg / kg, approximately 425 μg / kg, approximately 450 μg / kg, approximately 475 μg / kg, approximately 500 μg / kg, approximately 525 μg / kg, approximately 550 μg / kg, approximately 575 μg / kg, approximately 600 μg / kg, approximately 625 μg / kg, approximately 650 μg / kg Approximately 675 μg / kg, approximately 700 μg / kg, approximately 725 μg / kg, approximately 750 μg / kg, approximately 775 μg / kg, approximately 800 μg / kg, approximately 825 μg / kg, approximately 850 μg / kg, approximately 875 μg / kg, approximately 900 μg / kg, approximately 925 μg / kg, approximately 950 μg / kg, approximately 975 μg / kg, 1 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg Administer at a dose of approximately 35 mg / kg, approximately 40 mg / kg, approximately 45 mg / kg, approximately 50 mg / kg, approximately 60 mg / kg, approximately 70 mg / kg, approximately 80 mg / kg, approximately 90 mg / kg, approximately 100 mg / kg, approximately 125 mg / kg, approximately 150 mg / kg, approximately 175 mg / kg, approximately 200 mg / kg, or any dose between the above doses, and administer daily at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) unit doses.

[0066] In some embodiments, the circular RNA or composition disclosed in this application may be administered continuously for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 28 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, or at least 50 days.

[0067] In some embodiments, the circular RNA or composition disclosed in this application may be administered for one or more treatment cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles), wherein each cycle lasts for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or at least 50 days; and the interval between each two cycles is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, two weeks, three weeks, or four weeks.

[0068] disease In one aspect, this application provides a method for treating a subject's disease, comprising administering to the subject a therapeutically effective amount of the circular RNA or composition disclosed in this application.

[0069] In one aspect, this application provides the use of the circular RNA or composition disclosed herein in the preparation of a medicament for treating a subject’s disease.

[0070] In one aspect, this application provides the circular RNA or composition disclosed herein for use in treating a subject's disease.

[0071] In some implementations, the disease is selected from: diabetes, type 1 diabetes, type 2 diabetes, malnutrition-associated diabetes, diabetic hyperosmolar hyperglycemic state, hypoglycemia in the context of diabetes, diabetic ketoacidosis, diabetic coma, uncontrolled and unstable diabetes.

[0072] In some implementations, the disease is selected from: hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, mixed hyperlipoproteinemia, hyperalpha lipoproteinemia, hypolipoproteinemia, hypoalpha lipoproteinemia, and hypobeta lipoproteinemia.

[0073] In some implementations, the disease is selected from: overweight, obesity, or specific nutritional excesses and malnutrition.

[0074] In some implementations, the disease is selected from: non-alcoholic fatty liver disease, non-alcoholic steatohepatitis.

[0075] In some implementation schemes, treating the disease is seen as reducing the risk of cardiovascular events.

[0076] In some implementation schemes, the subjects are adults with type 2 diabetes and a history of cardiovascular disease.

[0077] In some implementations, the disease is selected from: metabolic dysfunction-associated fatty liver disease (MAFLD) or steatohepatitis (MASH).

[0078] While the description of the pharmaceutical compositions provided in this application is primarily directed toward pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally also suitable for administration to any other animal, such as non-human animals, for example, non-human mammals.

[0079] Example To make the objectives and technical solutions of this application clearer, the application will be further described below with reference to specific embodiments. It should be understood that these embodiments are not intended to limit the scope of this application. Furthermore, specific experimental methods not mentioned in the following embodiments are all performed according to conventional experimental methods.

[0080] Example 1: Vector Construction [Amended on April 3, 2025, in accordance with PCT Rule 91] The coding sequences for different GLP-1 variants have been internally optimized. The optimized cDNA sequences (corresponding to SEQ ID NOs: 7-20, 22-28) were synthesized by Azenta Life Sciences. Typically, the synthesized fragments are cloned into the pUC57 plasmid via the Spe I / EcoRI site, as... Figure 1 As shown. BspQ I (Vazyme, DD4302-PC-02) was added and incubated at 50°C for 2 hours (h) to linearize the circular RNA plasmid.

[0081] Example 2: Production and purification of circular RNA Circular RNA precursors were synthesized using T7 RNA polymerase (Vazyme, DD4101-PC-03) with linearized circular RNA plasmids as templates via in vitro transcription (IVT). After IVT, the RNA product was treated with DNase I (Vazyme, DD4104-PC-01) at 37°C for 15 min to remove the DNA template, and then purified using an RNA purification kit (NEB, T2040L). The purified circular RNA precursors (SEQ ID NO: 7, 8, 9, 14, 15, 16) were ligated into circular RNA using T4 RNA ligase II (Kactus, TRL-BE103-C1) at 25°C for 2 h. After ligation and purification, the circular RNA product was treated with ribonuclease R (Novoprotein, GMP-E224-M001) at 37°C for 30 min to remove linear RNA. Figure 2 ).

[0082] Purified circular RNA was separated by denaturation on a urea-polyacrylamide gel electrophoresis gel (Beyotime, R0218S). In short, 200 ng of RNA was mixed with an equal volume of 2×RNA loading buffer (NEB, B0363S), heated at 70°C for 10 minutes, and then immediately placed on ice. For denaturing gel electrophoresis, ssRNA gradient standards (NEB, N0362S) were used as the RNA molecular weight standard. After electrophoresis (150V, 1 hour), the gel was stained with YeaRed nucleic acid gel dye (Yeason, 10203ES76), and the RNA bands were visualized using a ChemiDoc MP imaging system (BioRad).

[0083] Example 3: Transfection and Circular RNA Expression in Mammalian Cells 293T cells were cultured in a 37°C, 5% CO2 incubator and in basal DMEM medium (Gibco, C11965500BT) supplemented with 10% fetal bovine serum (Corning, 35-081-CV) and 1% penicillin / streptomycin (Invitrogen, 15140148). Cells were passaged every 2–3 days. Before transfection, 300,000 cells / well were seeded into 24-well plates. Next, 500 ng of ribonuclease R-treated circular RNA was transfected into the cells using Lipofectamine RNAiMAX (Invitrogen, 13778030).

[0084] Sixteen hours after transfection, cell supernatant was collected, and then Western blotting was performed to analyze protein expression. In short, the supernatant of GLP-1 variants (nucleotide sequences SEQ ID NO: 9, 14) was separated in a 4-12% gradient SDS-PAGE (120V, 120 min), and the protein was transferred to a PVDF membrane via semi-dry transfer (2.5V, 30 min). After blocking, the membrane was incubated with anti-human IgG-Fc (Sino Biological, SSA001) and then stained. Western blotting showed that the GLP-1 variant was secreted into the cell supernatant. Figure 3 ).

[0085] Example 4: Detection of GLP-1 receptor ERK phosphorylation 293T and 3T3-L1 cells were cultured in a 37°C, 5% CO2 incubator and in basal DMEM medium (Gibco, C11965500BT) supplemented with 10% fetal bovine serum (Corning, 35-081-CV) and 1% penicillin / streptomycin (Invitrogen, 15140148). Prior to stimulation, the expression plasmid encoding human GLP1R was transiently transfected into 293T and 3T3-L1 cells for 18 hours. After starving the 293T and 3T3-L1 cells for 3 hours, 300 μL of cell supernatant containing the GLP-1 variant (as described in Example 3 above) was added to the starved cells for stimulation at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h. At the corresponding time points, cells were collected and lysed in a buffer containing inhibitors of phosphatase and protease (Beyotime, P1045). Immunoblot analysis of cell lysates was performed using anti-phosphorylated ERK (CST, 4370T) and anti-ERK (CST, 9107S). 293T cells were stimulated with a GLP-1 variant encoded by circular RNA. Figure 4 A) and 3T3-L1 cells ( Figure 4 B) ERK phosphorylation can be induced in 15 minutes.

[0086] Example 5: In vitro detection of GLP-1 receptor luciferase reporter gene A luciferase reporter gene detection system was constructed by stably overexpressing human GLP1R and a reporter gene containing a cAMP response element (CRE) and luciferase in 293T cells. In this system, luciferase expression is regulated by endogenous cAMP signal transduction. 293T-GLP1R-luciferase cells were cultured in a cell culture incubator at 37°C and 5% CO2, and supplemented with 10% fetal bovine serum (Corning, 35-081-CV), 1% penicillin / streptomycin (Invitrogen, 15140148), 5 μg / mL blastomycin (Gibco, A1113903), and 2 μg / mL puromycin (Gibco, A1113803) in basal DMEM medium (Gibco, C11965500BT). As described in Example 3 above, 293T-GLP1R-luciferase cells were starved for 3 hours, followed by treatment with 300 μL of cell supernatant containing GLP-1. After 24 hours of treatment, firefly luciferase activity was monitored using a Steady-Glo luciferase detection system (Promega, E2520), and luminescence intensity was read using a Spark multi-well microplate reader (TECAN). The circular RNA-encoded GLP-1 variant binds to human GLP1R, activating cAMP and driving the expression of the luciferase reporter gene (…). Figure 5).

[0087] Example 6: Repeated-dose study in Western diet (WD) induced BKS-db / db mice SPF-grade male BKS-db / db mice (8–9 weeks old) (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were housed under a 12-hour light / 12-hour dark cycle with free access to food and water. Mice underwent a one-week acclimatization period before the study began. Based on blood glucose levels and body weight, mice were randomly assigned to three groups. Throughout the experimental period, BKS-db / db mice were fed a Western diet, which exacerbated typical disease symptoms of type 2 diabetes (T2D) and metabolic-associated fatty liver disease (MAFLD). Wild-type BKS mice maintained normal diet and water access. Mice were injected with specified doses of the test drugs (semaglutide, 10 nmol / kg, subcutaneous injection, every three days; circular RNA-GLP003, intravenous injection, 0.9 nmol / kg on day 0, 0.2 nmol / kg on day 7) or solvents. The circular RNA used here, GLP003, is a circular RNA composed of the CVB3-IRES sequence (SEQ ID NO: 6) and the coding sequence of circular RNA-GLP003 (SEQ ID NO: 9). Body weight was recorded twice weekly. Blood lipid changes were measured using a chemiluminescence analyzer (Hitachi). Results showed that the circular RNA treatment group was superior to the semaglutide treatment group in terms of weight loss. Figure 6 In addition, total cholesterol levels decreased significantly on day 14. Figure 7 ).

[0088] Example 7: Repeated-dose study of a high-fat diet-induced obesity model (DIO model) Male C57BL / 6J mice were housed under a 12-hour light / dark cycle and fed a high-fat diet for 12–14 weeks. After a one-week acclimatization period, the high-fat diet-induced mice were divided into three groups based on body weight and blood glucose levels. Subsequently, the animals were subcutaneously injected with either semaglutide (10 nmol / kg, every three days) or circular RNA-GLP003 (0.9 nmol / kg, once a week). Body weight and food consumption were measured twice a week. At the end of the study, body composition was determined using an MRI system (QMR-06-060H small animal body composition analyzer, Numai, China). Simultaneously, blood glucose concentration was measured after 5 hours of fasting. After 4 weeks of subcutaneous administration, circular RNA-GLP003 reduced the body weight of DIO mice, with an effect comparable to semaglutide treatment. Figure 8 According to reports, smegglutinin primarily demonstrates significant weight loss potential by reducing fat mass, but it also significantly induces muscle loss, a finding confirmed in DIO mice. However, the significant weight loss following treatment with circular RNA-GLP003 was due to a substantial decrease in fat mass, while the lean body mass as a percentage of total body weight remained unchanged. Figure 9 A and 9B). Circular RNA-GLP003 and smegglutinin can significantly inhibit food intake in DIO mice ( Figure 10 Furthermore, compared to solvents, circular RNA-GLP003 treatment significantly reduced fasting blood glucose concentrations. Figure 11 (p<0.0001). In particular, the reduction in blood glucose concentration by administration of circular RNA-GLP003 was greater than that by administration of smegglutide (p=0.023). Therefore, compared with smegglutide, the lower dosing frequency of circular RNA-GLP003 can better reduce fat mass while maintaining lean body mass.

[0089] Example 8: Activation of c-fos signal transduction in the mouse central nervous system Male C57BL / 6J mice were housed in a specific pathogen-free facility at Youshu Life Sciences Co., Ltd. To investigate brain regions activated by circular RNA-GLP003 (labeled with c-fos) to regulate appetite, mice were injected with semaglutide (10 nmol / kg, subcutaneously) 4 hours later or with circular RNA-GLP003 (0.9 nmol / kg, intravenously) 6 hours later. Brain tissue was collected, fixed overnight in 4% formalin at 4°C, followed by standard dehydration and paraffin embedding. Immunofluorescence staining was performed on sections to detect c-fos signal activation in different brain regions (Servicebio). Quantitative analysis of cFos signal intensity in each brain region was performed using Fiji software at 20x magnification.

[0090] Following administration of circular RNA-GLP003, increased c-fos activity was observed in the brainstem, including the organ of endplates (OV), subfornix (SFO), area post-apical (AP), dorsal motor nucleus of the vagus nerve (DMX), and nucleus of the solitary tract (NTS), as well as in the forebrain, including the bed nucleus of the stria terminalis (BST), lateral preoptic area (LPO), central amygdala (CeA), lateral hypothalamus (LHA), parathalamic nucleus (PSTN), dorsal midline thalamus group (MTN), and parabrachial nucleus (PB). Figure 12 (A and 12B). These brain regions have previously been reported to be associated with regulating food intake. Interestingly, the activation of c-fos signaling in the brain regions differed between treatments with semaglutide and circular RNA-GLP003. For example, circular RNA-GLP003 induced higher c-fos levels in the PB region, while semaglutide induced stronger c-fos activity in the BST region. This unique neural activation may be due to the different routes of administration and drug forms. Therefore, circular RNA-GLP003 activated c-fos signaling in mouse brain regions, thereby reducing appetite and further reducing weight.

[0091] Since the methods, compounds, and compositions of this application have been fully described, those skilled in the art will understand that the same operations can be performed under a wide range of equivalent conditions, formulations, and other parameters without affecting the scope of the methods, compounds, and compositions or any embodiments thereof provided in this application.

[0092] All patents, patent applications and publications cited in this application are incorporated herein by reference in their entirety.

[0093] sequence list Table 1: Serial Numbers Used in This Application

[0094] Amino acid sequence of wild-type GLP-1 (7-37) SEQ ID NO: 1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG The amino acid sequence of GLP-1-IgG1 Fc SEQ ID NO: 2 METDTLLLWVLLLWVPGSTGHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK amino acid sequence of GLP-1-IgG1 Fc (YTE) SEQ ID NO: 3 METDTLLLWVLLLWVPGSTGHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of GLP-1-IgG4 Fc SEQ ID NO: 4 METDTLLLWVLLLWVPGSTGHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Amino acid sequence of GLP-1- IgG4 Fc (YTE) SEQ ID NO: 5 METDTLLLWVLLLWVPGSTGHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO: 6 (CVB3-IRES) 1 TTAAAACAGC CTGTGGGTTG ATCCCACCCA CAGGCCCATT GGGCGCTAGC 51 ACTCTGGTAT CACGGTACCT TTGTGCGCCT GTTTTATACC CCCTCCCCCA 101 ACTGTAACTT AGAAGTAACA CACACCGATC AACAGTCAGC GTGGCACACC 151 AGCCACGTTT TGATCAAGCA CTTCTGTTAC CCCGGACTGA GTATCAATAG 201 ACTGCTCACG CGGTTGAAGG AGAAAGCGTT CGTTATCCGG CCAACTACTT 251 CGAAAAACCT AGTAACACCG TGGAAGTTGC AGAGTGTTTC GCTCAGCACT 301 ACCCCAGTGT AGATCAGGTC GATGAGTCAC CGCATTCCCC ACGGGCGACC 351 GTGGCGGTGG CTGCGTTGGC GGCCTGCCCA TGTTTGGGGA AACCCAAACA 401 TGGGACGCTC TAATACAGAC ATGGTGCGAA GAGTCTATTG AGCTAGTTGG 451 TAGTCCTCCG GCCCCTGAAT GCGGCTAATC CTAACTGCGG AGCACACACC 501 CTCAAGCCAG AGGGCAGTGT GTCGTAACGG GCAACTCTGC AGCGGAACCG 551 ACTACTTTGG GTGTCCGTGT TTCATTTTAT TCCTATACTG GCTGCTTATG 601 GTGACAATTG AGAGATCGTT ACCATATAGC TATTGGATTG GCCATCCGGT 651 GACTAATAGA GCTATTATAT ATCCCTTTGT TGGGTTTATA CCACTTAGCT 701 TGAAAGAGGT TAAAACATTA CAATTCATTG TTAAGTTGAA TACAGCA GLP-1-like 1-IgG4 Fc complexes SEQ ID NO: 7 1 ATGGAAACAG ACACGCTGCT GCTCTGGGTG CTGCTGCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTACC 101 TGGAGGAGCA GGCGGCCAAG GAGTTCATTG CCTGGCTGGT GAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCGGC 201 GGAGAGCAAA TATGGGCCGC CGTGCCCGCC GTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CTCGGTCTTC CTCTTCCCGC CCAAGCCCA GGACACCCTC 301 ATGATCAGCC GCACGCCGGA GGTCACCTGT GTGGTGGTGG ATGTTTCTCA 351 AGAAGATCCT GAAGTACAGT TCAACTGGTA TGTAGATGGT GTAGAAGTTC 401 ATAATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT TCTTCATCAG GACTGGCTAA ATGGAAAAGA 501 ATATAAATGT AAAGTCAGCA ACAAGGGCCT GCCCAGCAGC ATTGAAAAGA 551 CCATCTCCAA GGCCAAGGGC CAGCCCCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCCAGCC AAGAAGAAAT GACCAAGAAC CAAGTCAGCC TCACCTGCCT 651 GGTGAAGGGC TTCTACCCCT CGGACATTGC TGTGGAGTGG GAGAGCAATG 701 GGCAGCCGGA GAACAACTAC AAGACCACGC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAAT GTCTTCTCCT GCAGTGTCAT GCATGAGGCG CTGCACAACC 851 ACTACACGCA GAAAAGCCTC AGCCTCAGCC TGGGC GLP-1-2-IgG4 Fc SEQ ID NO: 8 1 ATGGAAACAG ACACGCTGCT GCTCTGGGTG CTGCTGCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTACC 101 TGGAGGAGCA GGCGGCCAAG GAGTTCATTG CCTGGCTGGT GAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGTTCTGC 201 AGAGAGCAAA TATGGGCCGC CCTGCCCGCC CTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CAGTGTCTTC CTCTTCCCGC CCAAGCCCA GGACACCCTC 301 ATGATCAGCC GCACGCCGGA GGTCACCTGT GTGGTGGTGG ATGTTTCTCA 351 AGAAGATCCT GAGGTGCAGT TCAACTGGTA TGTGGATGGT GTTGAAGTTC 401 ATATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TCCTCACGGT GCTGCACCAG GACTGGCTAA ATGGAAAAGA 501 ATATAAATGT AAAGTCAGCA ACAAGGGCCT GCCCAGCAGC ATTGAGAAGA 551 CCATCTCCAA GGCCAAGGGC CAGCCCCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCCAGCC CAGGTCAGCC CAGGTCAGCC TCACCTGCCT 651 GGTGAAGGGC TTCTACCCCA GTGACATTGC TGTGGAGTGG GAGAGCAATG 701 GCCAGCCTGA GAACAACTAC AAGACCACCC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 GROUP GTCTTCTCCT GCAGTGTCAT GCATGAGGCC CTGCACAACC 851 ACTACACCCA GAAAAGCCTC AGCCTCAGCC TGGGC GLP-1-like 3-IgG4 Fc complexes SEQ ID NO:9 1 ATGGAAACAG ACACGCTGCT GCTCTGGGTG CTGCTGCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTATT 101 AGCAGCAAAA GAATTTATTG CCTGGCTGGT GAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCTGC 201 AGAAAGTAAA TATGGGCCGC CCTGCCCGCC CTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CAGTGTCTTC CTCTTCCCGC CCAAGCCCA GGACACCCTC 301 ATGATTTCAA GAACTCCAGA AGTCACCTGT GTGGTGGTGG ATGTTTCTCA 351 AGAAGATCCT GAAGTTCAGT TCAACTGGTA TGTAGATGGT GTTGAAGTTC 401 ATATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT TTTACATCAA ATGGAAAAGA 501 ATATAAATGT AAAGTTTCAA ATAAAGGCCT GCCCAGCAGC ATTGAAAAAA 551 CCATCTCCAA AGCAAAAGGA CAGCCCCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCCAGCC AAGAAGAAAT GACCAAAAAT CAAGTTTCTC TCACCTGCCT 651 GGTGAAGGGC TTCTACCCCA GTGATATTGC TGTGGAGTGG GAGAGCAATG 701 GGCAGCCAGA AAATAATTAT AAAACCACCC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACT GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAAT GTCTTCTCCT GCAGTGTCAT GCATGAAGCC CTGCACAACC 851 ACTACACCCA GAAAAGCCTC AGCCTCAGCC TGGGC Coding sequence of GLP-1-variant 4-IgG4 Fc SEQ ID NO: 10 1 ATGGAGACGG ACACGCTGCT GCTCTGGGTC CTCCTCCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTACT 101 TGGAAGAACA AGCAGCAAAA GAATTTATTG CCTGGCTTGT AAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCGGC 201 GGAGAGCAAA TATGGGCCGC CGTGCCCGCC GTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CTCGGTCTTC CTCTTCCCGC CCAAGCCCAA GGACACGCTC 301 ATGATCAGCC GCACGCCGGA GGTCACCTGT GTTGTTGTAG ATGTTTCTCA 351 AGAAGATCCT GAAGTACAGT TCAACTGGTA TGTAGATGGT GTAGAAGTTC 401 ATATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT TCTTCATCAA GACTGGCTAA ATGGAAAAGA 501 ATATAAATGT AAAGTCAGCA ACAAGGGCCT GCCCAGCAGC ATTGAAAAAA 551 CCATCTCCAA AGCAAAAGGA CAGCCGCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCGTCCC FAST FAST CAAGTCAGCC TCACCTGCCT 651 TGTAAAAGGC TTCTACCCGT CGGACATTGC TGTGGAGTGG GAGAGCAATG 701 GGCAGCCGGA GAACAACTAC AAGACCACGC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 GROUP GTCTTCTCCT GCAGTGTCAT GCATGAGGCG CTGCACAACC 851 ACTACACGCA GAAGAGCCTC AGCCTCAGCC TGGGC GLP-1-like 5-IgG4 Fc complexes SEQ ID NO: 11 1 ATGGAGACGG ACACGCTGCT GCTCTGGGTC CTCCTCCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTACC 101 TAGAAGAACA AGCAGCAAAA GAATTTATTG CCTGGCTTGT AAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCGGC 201 GGAGAGCAAA TATGGGCCGC CGTGCCCGCC GTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CTCGGTCTTC CTCTTCCCGC CCAAGCCCAA GGACACGCTC 301 ATGATCAGCC GCACGCCGGA GGTCACCTGT GTTGTTGTAG ATGTTTCTCA 351 AGAAGATCCT GAAGTACAGT TCAACTGGTA TGTAGATGGT GTAGAAGTTC 401 ATAATGCCAA GACCAAGCCG CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT TCTTCATCAA GACTGGCTAA ATGGAAAAGA 501 ATATAAATGT AAAGTCAGCA ACAAAGGACT TCCCAGCAGC ATAGAAAAAA 551 CCATCTCCAA AGCAAAAGGA CAGCCGCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCGTCCC AAGAAGAAAT GACCAAGAAC CAAGTCAGCC TCACCTGCCT 651 TGTAAAAGGC TTCTACCCGT CGGACATTGC TGTAGAATGG GAGAGCAATG 701 GGCAGCCGGA GAACAACTAC AAGACCACGC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAAT GTCTTCTCCT GCAGTGTCAT GCATGAGGCG CTGCACAACC 851 ACTACACGCA GAAGAGCCTC AGCCTCTCGC TGGGC Coding sequence of GLP-1-variant 6-IgG4 Fc SEQ ID NO: 12 1 ATGGAAACAG ACACCCTGCT GCTGTGGGTG CTGCTGCTGT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTACC 101 TGGAGGAGCA GGCTGCCAAA GAATTTATTG CCTGGCTGGT GAAAGGAGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCTGC 201 AGAGAGCAAA TATGGGCCGC CCTGCCCGCC CTGCCCAGCC CCGGAGGCGG 251 CGGGGGGCCC CAGTGTCTTC CTCTTCCCGC CCAAGCCCAA GGACACCCTC 301 ATGATTTCAA GAACTCCAGA AGTCACCTGT GTGGTGGTGG ATGTTTCTCA 351 AGAAGATCCT GAAGTTCAGT TCAACTGGTA TGTGGATGGT GTTGAAGTTC 401 ATAATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT GCTGCACCAG GACTGGTTAA ATGGAAAAGA 501 ATATAAATGT AAAGTTTCAA ATAAAGGCCT GCCCAGCAGC ATTGAAAAAA 551 CCATCAGCAA AGCAAAAGGA CAGCCCCGGG AGCCCCAGGT GTACACGCTG 601 CCGCCCAGCC AGAAGAAAT GACCAAAAAT CAAGTCAGCC TCACCTGCCT 651 GGTGAAGGGC TTCTACCCCA GTGATATTGC TGTGGAGTGG GAGAGCAATG 701 GGCAGCCAGA AAATAATTAT AAAACCACCC CACCTGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACT GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAT GTCTTCAGCT GCAGTGTCAT GCATGAAGCC CTGCACAACC 851 ACTACACCCA GAAGAGCCTC AGCCTCAGCC TGGGC GLP-1-like 7-IgG4 Fc complexes SEQ ID NO: 13 1 ATGGAAACAG ACACGCTGCT GCTCTGGGTT TATTATTAT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CTCAGATGTT TCTTCATATT 101 AGCAGCAAAA GAATTTATTG CCTGGCTTGT AAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGTTCTGC 201 AGAAAGTAAA TATGGGCCGC CCTGCCCGCC CTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CAGTGTCTTC CTCTTCCCGC CCAAGCCCAA AGATACTTTA 301 ATGATCAGCC GCACGCCGGA GGTCACCTGT GTTGTTGTAG ATGTTTCTCA 351 AGAAGATCCT GAAGTTCAGT TCAACTGGTA TGTAGATGGT GTTGAAGTTC 401 ATAATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT TTTACATCAA GACTGGTTAA ATGGAAAAGA 501 ATATAAATGT AAAGTTTCAA ATAAAGGCCT GCCCAGCAGC ATTGAA AAAA 551 CCATCTCCAA AGCAAAAGGA CAGCCCCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCCAGCC AAGAAGAAAT GACCAAAAAT CAAGTTTCTC TCACCTGCCT 651 TGTAAAAGGC TTCTACCCCA GTGATATTGC TGTGGAGTGG GAATCAAATG 701 GGCAGCCAGA AAATAATTAT AAAACCACGC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAAT GTCTTCTCCT GCAGTGTCAT GCATGAAGCC CTGCACAACC 851 ACTACACCCA GAAGAGCCTC AGCCTCAGCC TGGGC Coding sequence of GLP-1-variant 1-IgG4 Fc (YTE) SEQ ID NO: 14 1 ATGGAAACAG ACACGCTGCT GCTCTGGGTG CTGCTGCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAG GCACCTTCAC CAGTGATGTC AGCAGCTACC 101 TGGAGGAGCA GGCGGCCAAG GAGTTCATTG CCTGGCTGGT GAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGTTCTGC 201 AGAGAGCAAA TATGGGCCGC CCTGCCCGCC CTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCC CAGTGTCTTC CTCTTCCCGC CCAAGCCCAA GGACACCCTC 301 TACATCACCC GGGAGCCAGA AGTCACCTGT GTGGTGGTGG ATGTTTCTCA 351 AGAAGATCCT GAGGTGCAGT TCAACTGGTA TGTGGATGGT GTAGAAGTTC 401 ATAATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TCCTCACGGT GCTGCACCAG GACTGGCTAA ATGGAAAAGA 501 ATATAAATGT AAAGTCAGCA ACAAGGGCCT GCCCAGCAGC ATTGAGAAGA 551 CCATCTCCAA GGCCAAGGGC CAGCCCCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCCAGCC AGGAGGAGAT GACCAAGAAC CAGGTCAGCC TCACCTGCCT 651 GGTGAAGGGC TTCTACCCCA GTGACATTGC TGTGGAGTGG GAGAGCAATG 701 GGCAGCCTGA GAACAACTAC AAGACCACCC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAT GTCTTCAGCT GCAGTGTCAT GCATGAGGCG CTGCACAACC 851 ACTACACCCA GAAAAGCCTC AGCCTCAGCC TGGGC GLP-1-complex2-IgG4 Fc (YTE antibody complex) . SEQ ID NO:15 1 ATGGAAACAG ACACGCTGCT GCTCTGGGTG CTGCTGCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTACC 101 TGGAGGAGCA GGCGGCCAAG GAGTTCATTG CCTGGCTGGT GAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCGGC 201 GGAGAGCAAA TATGGGCCGC CGTGCCCGCC GTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CTCGGTCTTC CTCTTCCCGC CCAAGCCCA GGACACGCTC 301 TACATCACCC GGGAGCCGGA GGTCACCTGT GTGGTGGTGG ATGTTTCTCA 351 AGAAGATCCT GAAGTACAGT TCAACTGGTA TGTAGATGGT GTAGAAGTTC 401 ATATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT TCTTCATCAG GACTGGCTAA ATGGAAAAGA 501 ATATAAATGT AAAGTCAGCA ACAAGGGCCT GCCCAGCAGC ATTGAAAAAA 551 CCATCTCCAA GGCCAAGGGC CAGCCCCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCCAGCC AGAAGAAAT CAAGTCAGCC TCACCTGCCT 651 GGTGAAGGGC TTCTACCCCT CGGACATTGC TGTGGAGTGG GAGAGCAATG 701 GGCAGCCGGA GAACAACTAC AAGACCACGC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 GROUP GTCTTCTCCT GCAGTGTCAT GCATGAGGCG CTGCACAACC 851 ACTACACGCA GAAAAGCCTC AGCCTCAGCC TGGGC Structure of GLP-1-like 3-IgG4 Fc (YTE) complex SEQ ID NO: 16 1 ATGGAAACAG ACACGCTGCT GCTCTGGGTG CTGCTGCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTATT 101 AGCAGCAAAA GAATTTATTG CCTGGCTGGT GAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCTGC 201 AGAAAGTAAA TATGGGCCGC CCTGCCCGCC CTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CAGTGTCTTC CTCTTCCCGC CCAAGCCCAA GGACACCCTC 301 TACATCACCC GGGAGCCAGA AGTCACCTGT GTGGTGGTGG ATGTTTCTCA 351 AGAAGATCCT GAAGTTCAGT TCAACTGGTA TGTAGATGGT GTTGAAGTTC 401 ATAATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT TTTACATCAA GACTGGTTAA ATGGAAAAGA 501 ATATAAATGT AAAGTTTCAA ATAAAGGCCT GCCCAGCAGC ATTGAAAAAA 551 CCATCTCCAA AGCAAAAGGA CAGCCCCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCCAGCC AAGAAGAAAT GACCAAAAAT CAAGTCAGCC TCACCTGCCT 651 GGTGAAAGGC TTCTACCCCA GTGATATTGC TGTGGAGTGG GAGAGCAATG 701 GGCAGCCAGA AAATAATTAT AAAACCACCC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAAT GTCTTCTCCT GCAGTGTCAT GCATGAAGCC CTGCACAACC 851 ACTACACCCA GAAAAGCCTC AGCCTCAGCC TGGGC Structure of GLP-1-like 4-IgG4 Fc (YTE) complex SEQ ID NO: 17 1 ATGGAGACCG ACACCCTGCT GCTGTGGGTG CTGCTGCTGT GGGTGCCTGG 51 CAGCACCGGC CACGGCGAGG GCACCTTCAC CAGCGACGTG AGCAGCTACC 101 TGGAGGAGCA GGCCGCCAAG GAGTTCATCG CCTGGCTGGT GAAGGGCGGC 151 GGCGGCGGCG GCGGCAGCGG CGGCGGCGGC AGCGGCGGCG GCGGCAGCGGC 201 TACGGCCCTC CTTGCCCTCC TTGCCCTGCC CCTGAGGCCG 251 CCGGCGGCCC TAGCGTGTTC CTGTTCCCTC CTAAGCCTA GGACACCCTG 301 TACATCACCA GAGAGCCTGA GGTGACCTGC GTGGTGGTGG ACGTGAGCCA 351 GGAGGACCCT GAGGTGCAGT TCAACTGGTA CGTGGACGGC GTGGAGGTGC 401 ACAACGCCAA GACCAAGCCT AGAGAGC AGTTCAACAG CACCTACAGA 451 GTGGTGAGCG TGCTGACCGT GCTGCACCAG GACTGGCTGA ACGGCAAGGA 501 GTACAAGTGC AAGGTGAGCA ACAAGGGCCT GCCTAGCAGC ATCGAGAAGA 551 CCATCAGCAA GGCCAAGGGC CAGCCTAGAG AGCCTCAGGT GTACACCCTG 601 CCTCCTAGCC GACCAAGAAC CAGGTGAGCC TGACCTGCCT 651 GGTGAAGGGC TTCTACCCTA GCGACATCGC CGTGGAGTGG GAGAGCAACG 701 GCCAGCCTGA GAACAACTAC AAGACCACCC CTCCTGTGCT GGACAGCGAC 751 GGCAGCTTCT TCCTGTACAG CAGACTGACC GTGGACAAGA GCAGATGGCA 801 GGAGGGCAAC GTGTTCAGCT GCAGCGTGAT GCACGAGGCC CTGCACAACC 851 ACTACACCCA GAAAAGCCTG AGCCTGAGCC TGGGC GLP-1-including 5-IgG4 Fc (YTE) complex SEQ ID NO: 18 1 ATGGAGACGG ACACGCTGCT GCTCTGGGTC CTCCTCCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTACC 101 AGCAGCAAAA GAATTTATTG CCTGGCTTGT AAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCGGC 201 GGAGAGCAAA TATGGGCCGC CGTGCCCGCC GTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CTCGGTCTTC CTCTTCCCGC CCAAGCCCA GGACACGCTC 301 TACATCACCC GGGAGCCGGA GGTCACCTGT GTTGTTGTAG ATGTTTCTCA 351 AGAAGATCCT GAAGTACAGT TCAACTGGTA TGTAGATGGT GTAGAAGTTC 401 ATAATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT TCTTCATCAA GACTGGCTAA ATGGAAAAGA 501 ATATAAATGT AAAGTCAGCA ACAAGGGCCT GCCCAGCAGC ATTGAAAAAA 551 CCATCTCCAA GGCCAAGGGC CAGCCGCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCGTCCC AAGAAGAAAT GACCAAGAAC CAAGTCAGCC TCACCTGCCT 651 TGTAAAAGGC TTCTACCCGT CGGACATTGC TGTGGAGTGG GAGAGCAATG 701 GGCAGCCGGA GAACAACTAC AAGACCACGC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAAT GTCTTCTCCT GCAGTGTCAT GCATGAGGCG CTGCACAACC 851 ACTACACGCA GAAGAGCCTC AGCCTCTCGC TGGGC Coding sequence of GLP-1-variant 6-IgG4 Fc (YTE) SEQ ID NO: 19 1 ATGGAGACGG ACACGCTGCT GCTCTGGGTC CTCCTCCTCT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTACC 101 TAGAAGAACA AGCAGCAAAA GAATTTATTG CCTGGCTTGT AAAAGGTGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCGGC 201 GGAGAGCAAA TATGGGCCGC CGTGCCCGCC GTGCCCGGCG CCGGAGGCGG 251 CGGGGGGCCC CTCGGTCTTC CTCTTCCCGC CCAAGCCCAA GGACACGCTC 301 TACATCACGC GGGAGCCGGA GGTCACCTGT GTTGTTGTAG ATGTTTCTCA 351 AGAAGATCCT GAAGTACAGT TCAACTGGTA TGTAGATGGT GTAGAAGTTC 401 ATAATGCCAA GACCAAGCCG CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT TCTTCATCAA GACTGGCTAA ATGGAAAAGA 501 ATATAAATGT AAAGTCAGCA ACAAAGGACT TCCCAGCAGC ATAGAAAAAA 551 CCATCTCCAA AGCAAAAGGA CAGCCGCGGG AGCCCCAGGT CTACACGCTG 601 CCGCCGTCCC AAGAAGAAAT GACCAAGAAC CAAGTCAGCC TCACCTGCCT 651 TGTAAAAGGC TTCTACCCGT CGGACATTGC TGTAGAATGG GAGAGCAATG 701 GGCAGCCGGA GAACAACTAC AAGACCACGC CGCCGGTGCT GGACAGTGAT 751 GGCTCCTTCT TCCTCTACAG CCGCCTCACG GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAAT GTCTTCTCCT GCAGTGTCAT GCATGAGGCG CTGCACAACC 851 ACTACACGCA GAAGAGCCTC AGCCTCTCGC TGGGC Coding sequence of GLP-1-variant 7-IgG4 Fc (YTE) SEQ ID NO: 20 1 ATGGAAACAG ACACCCTGCT GCTGTGGGTG CTGCTGCTGT GGGTTCCTGG 51 CAGCACTGGA CATGGAGAAG GCACCTTCAC CAGTGATGTC AGCAGCTACC 101 TGGAGGAGCA GGCGGCCAAA GAATTTATTG CCTGGCTGGT GAAAGGAGGT 151 GGTGGTGGTG GTGGCAGTGG TGGTGGTGGC AGTGGTGGTG GTGGCTCTGC 201 AGAGAGCAAA TATGGGCCGC CCTGCCCGCC CTGCCCGGCC CCGGAGGCGG 251 CGGGGGGCCC CAGTGTCTTC CTCTTCCCGC CCAAGCCCAA GGACACCCTC 301 TACATCACCC GGGAGCCAGA AGTCACCTGT GTGGTGGTGG ATGTTTCTCA 351 AGAAGATCCT GAAGTTCAGT TCAACTGGTA TGTGGATGGT GTTGAAGTTC 401 ATAATGCCAA GACCAAGCCC CGGGAGGAGC AGTTCAACAG CACCTACAGA 451 GTTGTTTCTG TTTTAACTGT GCTGCACCAG GACTGGCTGA ATGGAAAAGA 501 ATATAAATGT AAAGTTTCAA ATAAAGGCCT GCCCAGCAGC ATTGAAAAAA 551 CCATCAGCAA AGCAAAAGGA CAGCCCCGGG AGCCCCAGGT GTACACGCTG 601 CCGCCCAGCC AAGAAGAAAT GACCAAAAAT CAAGTCAGCC TCACCTGCCT 651 GGTGAAGGGC TTCTACCCCA GTGATATTGC TGTGGAGTGG GAGAGCAATG 701 GGCAGCCAGAAAAATTATAAAACCACCCACCTGTGCTGGACAGTGAT 751 GGCTCCTTCTTCCTCTACAG CCGCCTCACT GTGGACAAGA GCCGCTGGCA 801 AGAAGGAAAT GTCTTCAGCT GCAGTGTCAT GCATGAAGCC CTGCACAACC 851 ACTACACCCA GAAGAGCCTC AGCCTCAGCC TGGGC The amino acid sequence of the GLP-1 variant SEQ ID NO: 21 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG GLP-1 Variant 1 coding sequence SEQ ID NO: 22 1 CATGGAGAAGGCACCTTCAC CAGTGATGTC AGCAGCTACC TGGAGGAGCA 51 GGCGGCCAAG GAGTTCATTG CCTGGCTGGT GAAAGGTGGT GGT GLP-1 variant 2 coding sequence SEQ ID NO: 23 1 CATGGAGAAGGCACCTTCAC CAGTGATGTC AGCAGCTACC TGGAGGAGCA 51 GGCGGCCAAG GAGTTCATTG CCTGGCTGGT GAAAGGTGGT GGT The coding sequence of GLP-1 variant 3 SEQ ID NO: 24 1 CATGGAGAAGGCACCTTCAC CAGTGATGTC AGCAGCTATT TAGAAGAACA 51 AGCAGCAAAA GAATTTATTG CCTGGCTGGT GAAAGGTGGT GGT The coding sequence of GLP-1 variant 4 SEQ ID NO: 25 1 CATGGAGAAGGCACCTTCAC CAGTGATGTC AGCAGCTACTTGGAAGAACA 51 AGCAGCAAAA GAATTTATTG CCTGGCTTGT AAAAGGTGGT GGT The coding sequence of GLP-1 variant 5 SEQ ID NO: 26 1 CATGGAGAAGGCACCTTCAC CAGTGATGTC AGCAGCTACC TAGAAGAACA 51 AGCAGCAAAA GAATTTATTG CCTGGCTTGT AAAAGGTGGT GGT The coding sequence of GLP-1 variant 6 SEQ ID NO: 27 1 CATGGAGAAGGCACCTTCAC CAGTGATGTC AGCAGCTACC TGGAGGAGCA 51 GGCTGCCAAA GAATTTATTG CCTGGCTGGT GAAAGGAGGT GGT The coding sequence of GLP-1 variant 7 SEQ ID NO: 28 1 CATGGAGAAGGCACCTTTCACCTCAGATGTTTCTTCATATTTAGAAGAACA 51 AGCAGCAAAA GAATTTATTG CCTGGCTTGT AAAAGGTGGT GGT amino acid sequence of signal peptide SEQ ID NO: 29 METDTLLLWVLLLWVPGSTG Amino acid sequence of IgG 1 Fc SEQ ID NO: 30 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of IgG 1 Fc (YTE) SEQ ID NO: 31 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of IgG 4 Fc SEQ ID NO: 32 AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Amino acid sequence of IgG 4 Fc (YTE) SEQ ID NO: 33 AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Amino acid sequence of the linker SEQ ID NO: 34 GGGGSGGGGSGGGGS

Claims

1. A circular RNA comprising a regulatory element and an expression element, wherein the expression element comprises a polynucleotide encoding GLP-1 or a variant thereof.

2. The circular RNA according to claim 1, wherein the regulatory element is an internal ribosome entry site (IRES) or a fragment thereof.

3. The circular RNA according to claim 2, wherein the IRES is selected from: Coxsackievirus B3 (CVB3) IRES, Enterovirus 71 (EV71) IRES, Encephalomyocarditis Virus (EMCV) IRES, Picornavir Virus (PV) IRES, Hepatitis C Virus (HCV) IRES, Adenovirus (AdV) IRES, Human Papillomavirus 31 (HPV31) IRES, Human Herpesvirus (HHV) IRES, Raul's Sarcoma Virus (RSV) IRES, Classical Swine Fever Virus (CSFV) IRES, FGF9 IRES, SLC7A1 IRES, and RUNX1 IRES, preferably the IRES is CVB3 IRES.

4. The circular RNA according to claim 1, wherein the polynucleotide encoding GLP-1 or a variant thereof has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 22, 23, 24, 25, 26, 27, or 28, preferably the polynucleotide encoding GLP-1 or a variant thereof as shown in SEQ ID NO: 22, 23, 24, 25, 26, 27, or 28.

5. The circular RNA according to claim 1, wherein the amino acid sequence of GLP-1 or a variant thereof is as shown in SEQ ID NO:

21.

6. The circular RNA of claim 1, wherein the expression element comprises a polynucleotide encoding Fc.

7. The circular RNA of claim 6, wherein the expression element comprises a polynucleotide encoding a linker between GLP-1 or a variant thereof and Fc.

8. The circular RNA according to claim 7, wherein the adapter is GGGGSGGGGSGGGGS (SEQ ID NO:34).

9. The circular RNA of claim 7, comprising, in the following order from 5' to 3': a polynucleotide encoding GLP-1 or a variant thereof, a polynucleotide encoding an adapter, and a polynucleotide encoding Fc.

10. The circular RNA of claim 8, wherein the Fc comprises one or more mutations.

11. The circular RNA of claim 8, wherein the Fc contains a YTE mutation.

12. The circular RNA according to claim 8, wherein the Fc is selected from: IgG1 Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc, preferably the Fc is IgG1 Fc or IgG4 Fc.

13. The circular RNA according to claim 6, wherein the amino acid sequence of Fc is as shown in SEQ ID NO: 30, 31, 32 or 33.

14. The circular RNA of claim 1, comprising, in the following order from 5' to 3': a polynucleotide encoding a signal peptide and a polynucleotide encoding GLP-1 or a variant thereof.

15. The circular RNA according to claim 14, wherein the amino acid sequence of the signal peptide is shown in SEQ ID NO:

29.

16. The circular RNA as claimed in any of the preceding claims, wherein the expression element comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with a sequence shown in any of SEQ ID NOs: 7-20, preferably the expression element comprises a sequence shown in any of SEQ ID NOs: 7-20, more preferably the expression element comprises a sequence shown in SEQ ID NO:

9.

17. A composition comprising the circular RNA of any one of the preceding claims, wherein the composition further comprises a pharmaceutically acceptable excipient.

18. The composition of claim 17, wherein the composition comprises nanoparticles, such as lipid nanoparticles.

19. A method of treating a subject's disease, comprising administering to the subject a therapeutically effective amount of the circular RNA of any one of claims 1-16, or administering to the subject the composition of claim 17 or claim 18.

20. Use of the circular RNA according to any one of claims 1-16 or the composition according to claim 17 or claim 18 in the preparation of a medicament for treating a subject's disease.

21. The circular RNA according to any one of claims 1-16 or the composition according to claim 17 or claim 18, for treating a disease in a subject.

22. The method or use according to any one of claims 19-21, wherein the disease is selected from: diabetes, type 1 diabetes, type 2 diabetes, malnutrition-associated diabetes, diabetic hyperosmolar hyperglycemic state, hypoglycemia in the context of diabetes, diabetic ketoacidosis, diabetic coma, uncontrolled and unstable diabetes.

23. The method or use according to any one of claims 19-21, wherein the disease is selected from: hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia, hyperalpha lipoproteinemia, hypolipoproteinemia, hypoalpha lipoproteinemia, and hypobeta lipoproteinemia.

24. The method or use according to any one of claims 19-21, wherein the disease is selected from: overweight, obesity, or specific nutritional excesses and malnutrition.

25. The method or use according to any one of claims 19-21, wherein the disease is selected from: non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH).

26. The method or use according to any one of claims 19-21, wherein treating the disease is reducing the risk of cardiovascular events.

27. The method or use according to claim 26, wherein the subject is an adult with type 2 diabetes and a confirmed cardiovascular disease.

28. The method or use according to any one of claims 19-21, wherein the disease is selected from: metabolic dysfunction-associated fatty liver disease (MAFLD) or steatohepatitis (MASH).

29. A method for reducing the weight of a subject, comprising administering to the subject a therapeutically effective amount of the circular RNA of any one of claims 1-16, or administering to the subject the composition of claim 17 or claim 18, wherein the method does not reduce the subject's lean muscle mass.

30. A method for reducing fat in a subject, comprising administering to the subject a therapeutically effective amount of the circular RNA of any one of claims 1-16, or administering to the subject the composition of claim 17 or 18, wherein the method does not reduce the subject's lean muscle mass.

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