Circular RNA molecule and use thereof

Abstract

The present invention provides a recombinant protein comprising a GLP-1 receptor agonist, an expression vector comprising a linear precursor RNA for expressing the recombinant protein, a circular RNA molecule, a composition comprising the circular RNA molecule, and a use thereof in the preparation of a drug for treating a metabolic disease.

Description

A circular RNA molecule and its applications Technical Field

[0001] This invention belongs to the field of gene-engineered drugs, specifically relating to a circular RNA molecule and its corresponding uses. Background Technology

[0002] In the treatment of diabetes and metabolic diseases, glucagon-like peptide-1 receptor (GLP-1R) agonists such as exenatide... Due to their significant effects in lowering blood sugar levels, delaying gastric emptying, and suppressing appetite, exenatide has become a highly regarded treatment option. For example, exenatide is a peptide hormone composed of 39 amino acids, with a structure similar to the naturally occurring peptide hormone GLP-1, and it exerts its effects by activating GLP-1R. By promoting insulin secretion and inhibiting glucagon release, exenatide helps improve glycemic control in patients with type 2 diabetes. However, like other peptide drugs or GLP-1 receptor agonist proteins, exenatide has limited stability and half-life in the human body, often requiring frequent dosing to maintain its efficacy.

[0003] Traditional protein drug therapy faces several major challenges. First, exenatide, due to its short peptide chain, is easily degraded by proteases, leading to a rapid loss of drug activity. Second, injectable protein drugs have short half-lives, requiring frequent injections, increasing treatment complexity and patient discomfort.

[0004] Circular RNA (circRNA) technology, as an emerging molecular tool, offers a novel solution for protein drug expression. circRNAs possess a unique circular structure, exhibiting greater stability and resistance to exonuclease degradation compared to linear mRNAs. This significantly prolongs their half-life in vivo, thereby reducing the frequency of dosing. Furthermore, circRNAs can enhance protein expression efficiency through rolling circle translation. By optimizing the sequence of circRNAs and inserting introns, splicing enhancers, or translation initiation regulatory elements, circRNAs can achieve long-term, stable expression of target proteins, making them particularly suitable for small peptide drugs like exenatide that require frequent dosing.

[0005] Circular RNA exists in a closed circular form, lacking the common 5' cap and 3' tail structures. Due to this circular structure, circular RNA can avoid degradation by exonucleases (such as RNase R), thus making it more stable in cells compared to linear RNA. However, precisely because of the lack of a 5' cap, protein translation of circular RNA relies on internal ribosome entry sites (IRES elements) to initiate translation. Wesselhoeft et al. successfully prepared large fragments of circular RNA using the AnaPIE circularization system constructed with type I ribozymes and achieved efficient protein translation by introducing IRES elements into the circular RNA (Wesselhoeft, Kowalski et al. 2018).

[0006] Invention Overview

[0007] Based on the above technical problems, in this invention, we designed a circular RNA molecule and developed its uses.

[0008] This invention relates to a recombinant protein comprising a GLP-1 receptor agonist selected from at least one of SEQ ID NO: 17, 19, 23-26, and a sequence having at least 90% identity to achieve the same function, and wherein one or more amino acid residues are substituted, added, inserted, or deleted in the aforementioned sequence; optionally, the recombinant protein comprises a signal peptide; optionally, the recombinant protein comprises a self-cleaving peptide selected from any one of SEQ ID NO: 20-22.

[0009] In one specific embodiment, the recombinant protein may be as shown in SEQ ID NO:3 or 16, or a sequence that achieves the same function with more than 90% identity, or a sequence in which one or more amino acid residues are substituted, added, inserted or deleted.

[0010] The present invention further relates to nucleic acid molecules capable of expressing the aforementioned recombinant proteins; preferably, the nucleic acid molecule may be a DNA molecule or an RNA molecule; optionally, the DNA molecule may be selected from SEQ ID NO:1, 4, 6, 8, 10, 12, 14, the RNA molecule may be selected from SEQ ID NO:2, 5, 7, 9, 11, 13, 15, and sequences that achieve the same function with more than 90% identity, and sequences in which one or more amino acid residues are substituted, added, inserted or deleted, preferably 1-3 residues.

[0011] In one specific embodiment, the present invention also relates to an expression vector capable of producing the aforementioned recombinant protein and containing the aforementioned nucleic acid molecule. The linear precursor RNA molecule includes homologous arms, exons, introns, spacer regions, internal ribosome entry sites, and a coding region; furthermore, the structure can be...

[0012] 5' outer homologous arm - IG5 - 5' intron - 3' intron - E2 - 5' spacer region - internal ribosome entry site (IRES) - coding region - 3' spacer region - E1 - IG3 - 3' outer

[0013] Homologous Arm

[0014] or

[0015] 5' outer homologous arm - 3' intron - 5' spacer region - internal ribosome entry site (IRES) - coding region - 3' spacer region - 5' intron - 3' outer homologous arm.

[0016] In one specific embodiment, it may include a circulatory element with RNA sequence SEQ ID NO: 27-37, or a circulatory element with RNA sequence SEQ ID NO: 49-55; further, corresponding circulatory elements with DNA sequence SEQ ID NO: 38-48, or circulatory elements with DNA sequence SEQ ID NO: 56-62, any one of which, as well as sequences that achieve the same function with more than 90% identity, and substitution, addition, insertion, or deletion of one or more amino acid residues in the above sequences.

[0017] In one specific embodiment, it can be the linear precursor RNA molecule shown in SEQ ID NO: 63-69, the DNA molecule shown in SEQ ID NO: 70-76 expressing the linear precursor RNA molecule, any one of the sequences that has more than 90% identity to achieve the same function, and the substitution, addition, insertion or deletion of one or more nucleotide residues in the above sequences.

[0018] In one specific embodiment, the expression vector is preferably a circular RNA molecule, the sequence of which may be as shown in SEQ ID NO: 77-83, or any one of the sequences that achieves the same function with more than 90% identity, or one or more nucleotide residues may be substituted, added, inserted or deleted in the above sequences.

[0019] The present invention further relates to a host cell capable of containing the aforementioned expression vector.

[0020] The present invention further relates to a composition comprising the aforementioned circular RNA molecule; preferably, the composition comprises lipids encapsulating the circular RNA molecule; the lipids comprise LNPs, liposomes, or exosome delivery systems.

[0021] LNP contains cationic or ionizable lipids selected from SM-102, ALC-0315, Dlin-MC3-DMA, DODMA, C12-200, DlinDMA, and HL1001; auxiliary lipids selected from DSPC, DOPE, DOPC, DOPG, or DOPS; cholesterol or cholesterol derivatives; and PEG lipids selected from mPEG2000-DMG, DSG-PEG2000, and ALC-0519.

[0022] The LNP composition preferably has a molar ratio of SM102 and / or HL1001: cholesterol: DSPC: mPEG-DMG-2K of (25-65): (25-70): (5-25): (0.5-5), more preferably (23-61): (27-66): (8-22): (0.7-4), or further, (27-58): (30-60): (8-19): (0.7-3.5), and more preferably (30-55): (30-50): (8-16): (0.7-2.0).

[0023] The concentration of the lipid solution can be between 5 mg / mL and 15 mg / mL, specifically 5-10 mg / mL, more preferably 5 mg / mL, 7 mg / mL, 10 mg / mL, 12 mg / mL, or 15 mg / mL, and even more preferably selected from 7 mg / mL, 10 mg / mL, or 12 mg / mL.

[0024] The present invention further relates to the use of recombinant proteins, nucleic acid molecules, expression vectors, host cells, and compositions in the treatment and prevention of metabolic diseases, and in the preparation of medicaments for the treatment of metabolic diseases.

[0025] This invention further relates to the use of recombinant proteins, nucleic acid molecules, expression vectors, host cells, and compositions in assisting weight loss, as well as their use in preparing foods and health products with weight loss functions.

[0026] This invention further relates to the use of recombinant proteins, nucleic acid molecules, expression vectors, host cells, and compositions in regulating insulin, blood glucose, cholesterol, and triglyceride levels in patients. The metabolic diseases mentioned include diabetes, obesity, dyslipidemia, elevated glucose levels, elevated insulin levels, and diabetic nephropathy.

[0027] definition

[0028] The term "circular nucleic acid molecule"

[0029] A closed, circular nucleic acid molecule. In some specific embodiments, the circular nucleic acid molecule is a circular RNA molecule. More specifically, the circular nucleic acid molecule is a circular mRNA molecule. The term "linear precursor RNA" refers to a circular RNA precursor capable of forming circular RNA through a circularization reaction, which is generally transcribed from a linear DNA molecule (e.g., a vector containing recombinant nucleic acid molecules).

[0030] The term "Type I intron"

[0031] Group I introns are ribozyme systems capable of self-splicing in the presence of GTP and Mg2+. Essentially, they are RNA nucleic acid sequences capable of self-cleavage and are widely distributed across many species, primarily involved in the catalytic cleavage of mRNA, tRNA, and rRNA precursors. The secondary structure of a Group I intron mainly consists of 10 arm-like structures (P1-P10) and 10 loop-like structures (L1-L10), along with connecting structures between the arms and loops. In this invention, introns include, but are not limited to, 5' Group I introns and / or 3' Group I introns.

[0032] The term "translation initiation element"

[0033] In this invention, the translation initiation element can be any type of element capable of initiating the translation of a target polypeptide. In some embodiments, the translation initiation element is an element comprising any one or more of the following sequences: an IRES sequence, a 5'UTR sequence, a Kozak sequence, a sequence comprising m6A modification (N(6)methyladenosine modification), or a complementary sequence of ribosomal 18S rRNA. In other embodiments, the translation initiation element can also be any other type of cap-independent translation initiation element. In some embodiments according to circular RNA, the circular RNA further comprises an IRES sequence. In some implementations, the IRES sequence is the IRES sequence of Taura syndrome virus, Trichophyton meningovirus, Tyler encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, rice blast virus, reticuloendotheliosis virus, Forman poliovirus 1, Proutia virus, Stali enterovirus, Kashmir bee virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, equine rhinitis virus, and Ectropis obliqua. Picorna-like viruses, encephalomyocarditis virus (EMCV), Drosophila C virus, cruciferous plant viruses, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic ringspot virus, classical swine fever virus, human FGF2, human SFTPAI, human AMLI / RUNXI, Drosophila antennapedia, human AQP4, human ATIR, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF 1. IRES sequences of mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, salivary viruses, coronaviruses, paraenteroviruses, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian pyrovirus, radish wrinkle virus, aptamers of eIF4G, Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), human enterovirus EV71, encephalocarditis virus EMCV, PV virus, or CSFV virus.

[0034] The term "spacer"

[0035] As used herein, a “spacer” refers to any consecutive nucleotide sequence that: 1) predicts to avoid interfering with proximal structures, such as those from IRES, coding or non-coding regions, or introns; 2) is at least 7 nucleotides long (optionally not exceeding 100 nucleotides); 3) is located downstream and near a 3' intron fragment and / or upstream and near a 5' intron fragment; and / or 4) contains one or more of the following: a) an unstructured region at least 5 nt long; b) a region predicted to pair with a distal (i.e., non-adjacent) sequence at least 5 nt long, which includes another spacer; and / or c) a structured region at least 7 nt long, the extent of which is limited to the spacer sequence.

[0036] In some embodiments, the spacer sequence may be, for example, at least 10 nucleotides long, at least 15 nucleotides long, or at least 30 nucleotides long. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides long. In some embodiments, the spacer sequence is no longer than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides long.

[0037] The spacer sequence can be a polyA sequence, a polyA-C sequence, a polyC sequence, or a poly-U sequence, or it can be specifically modified according to the IRES. The spacer sequence described herein can have two functions: (1) promoting circularization and (2) promoting functionality by allowing introns and IRES to fold correctly. More specifically, the spacer sequence described herein is designed to have three priorities: 1) being inert to the folding of proximal intron and IRES structures; 2) adequately separating the secondary structures of introns and IRES; and 3) containing a spacer-spacer complementary region to promote the formation of a "splicing bubble". In one embodiment, the vector is compatible with many possible IRES and coding or non-coding regions as well as two spacer sequences.

[0038] The terms "homogeneous arm" and "external homogeneous arm" are also mentioned.

[0039] The 5' and 3' homologous arms can be synthetic sequences that differ from the internal homologous regions but have similar functions. The length of the homologous arms can be, for example, about 5-55 nucleotides, about 9-19 nucleotides, for example, about 5, about 10, about 20, about 30, about 40, or about 50 nucleotides. In another embodiment, the length of the homologous arm can be 9 nucleotides. In another embodiment, the length of the homologous arm can be 19 nucleotides. In some embodiments, the length of the homologous arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the length of the homologous arm does not exceed 50, 45, 40, 35, 30, 25, or 20 nucleotides. In some embodiments, the length of the homologous arms is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 nucleotides.

[0040] The term "exon fragment"

[0041] An exon is a segment of a gene that is retained after mRNA splicing; the vast majority of exons are coding sequences. The spliced ​​exon sequences form the mature mRNA encoded by the polypeptide chain. The sum of all exons in the genome is called the exome.

[0042] The term "encoding area"

[0043] Nucleic acid sequences that can be translated into amino acids and ultimately form proteins. More specifically, these are the nucleic acid sequences in messenger RNA molecules that can be translated into amino acids and ultimately form proteins.

[0044] The term "recombinant nucleic acid molecule"

[0045] Recombinant polynucleotides are polynucleotides with sequences that are not linked together in nature. These polynucleotides can be contained in suitable vectors, which can then be transformed into suitable host cells. The polynucleotides are then expressed in the recombinant host cells to produce, for example, "recombinant polypeptides," "recombinant proteins," or "fusion proteins."

[0046] The term "linking peptide"

[0047] This method is often used in multi-gene expression scenarios. Multiple genes are repeated within an expression vector, each linked by a connective peptide (self-cleaving peptide). During translation, the translated polypeptide chain contains three proteins, each separated by a self-cleaving peptide. Once translation is complete, the self-cleaving peptide promotes self-cleavage, releasing multiple independent proteins. This approach allows for the simultaneous expression of multiple proteins while ensuring they function independently. Examples of connective peptides include P2A, T2A, and E2A.

[0048] The term "cyclic system"

[0049] The circularization system is a linear RNA construct that can form a circular RNA molecule through circularization. Circularization systems including the AzoPIE circularization system (see PCT / CN2023 / 090879) and the FlexCirc circularization system (patent application number 202311808436.9) can be used, and the full text of the above patents is cited for reference in this patent regarding the linear RNA precursor structure.

[0050] The term "host cell"

[0051] Generally, this refers to the modification or recombination of the genetic material of host cells using genetic engineering or cell fusion techniques to obtain cells with stable and unique inherited traits. This encompasses host cells that differ from parental cells after the introduction of recombinant nucleic acid molecules, recombinant expression vectors, or circular RNA, and can be eukaryotic or prokaryotic cells. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the nucleic acids of this invention, such as DNA and / or RNA sequences, and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / 0 myeloma cells, HEK293T, HEK293, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), CoS cells, 3T3, NSO, HT-1080, PERC6, CAP, HKB-11, and Huh-7.

[0052] The terms "conversion, transfection, transduction"

[0053] Transformation, transfection, and transduction are, in the sense commonly understood by those skilled in the art, the process of introducing exogenous nucleic acids (DNA, RNA, etc.) into a host. These methods include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate (Ca3(PO4)2) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0054] The terms "identity," "homology," or "similarity"

[0055] When describing amino acid or nucleic acid sequences relative to a reference sequence, the percentage of identical amino acids or nucleotides between the two sequences is determined using conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0056] The term "identity" refers to at least 66%, and may be 66%, 67%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, with respect to any of the reference sequences herein, including all sequences involved in the claims or specification. All sequences include variations of the above-described concept of at least 66% identity.

[0057] "Complementarity" refers to the ability of nucleic acids to form hydrogen bonds through conventional Watson-Crick base pairing or other non-traditional types of base pairing. In the case of the nucleic acid molecules of this invention, the binding free energy of the nucleic acid molecule and its complementary sequence is sufficient to enable the relevant functions of the nucleic acid, such as RNAi activity. The determination of the binding free energy of nucleic acid molecules is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol., 1987, LII, pp. 123-133; Frier et al., PNAS, 1986, 83, 9373-9377; Turner et al., J Am. Chem. Soc, 1987, 109, 3783-3785). The complementarity percentage indicates the complementarity of a nucleic acid molecule with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides in total, representing 50%, 60%, 70%, 80%, 90%, and 100% respectively, in the first oligonucleotide that pairs with the bases of the second nucleic acid sequence having 10 nucleotides). "Perfect complementarity" means that all consecutive residues in the nucleic acid sequence will form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence.

[0058] As used herein, a "vector" refers to a segment of DNA that is synthetic (e.g., using PCR) or extracted from a virus, plasmid, or cell of a higher organism, into which a foreign DNA fragment may be inserted or has been inserted for cloning and / or expression purposes. In some embodiments, the vector can be stably maintained in an organism. A vector may contain, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, granules, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like.

[0059] The term "Exendin-4" refers to a 39-amino acid peptide and its active conjugates and fragments, which activate the GLP-1 (glucagon-like peptide-1) receptor, thereby increasing intracellular cAMP. Exendin-4 and Exenatide are different names for the same substance; Exenatide is a synthetic form of Exendin-4.

[0060] The terms “individual,” “patient,” or “subject” include (optionally added depending on whether therapeutic use is involved) mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0061] The term "treatment" refers to the process of exposing a subject to (e.g., administering medication) the circular RNA, circularized precursor RNA, and recombinant nucleic acid vectors and compositions of the present invention after contracting a disease, thereby alleviating the symptoms of the disease compared to when not exposed, without implying the necessity of completely suppressing the symptoms of the disease. Contracting a disease means that the body exhibits symptoms of a disease.

[0062] The term "prevention" means that, before the onset of a disease, by exposing the subject to (e.g., administering medication) the circular RNA, recombinant nucleic acid vector, composition, etc. of the present invention, the symptoms of the disease are reduced compared to when the subject is not exposed, and does not mean that the disease must be completely suppressed.

[0063] The term "effective amount" refers to the quantity or dose of the recombinant nucleic acid molecule, recombinant expression vector, circularized precursor RNA, circular RNA, vaccine, or composition of the present invention, which, when administered to a patient in a single or multiple doses, produces the desired effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0064] The term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining an active antibody or its antigen-binding fragment with a liquid carrier, a finely fragmented solid carrier, or both.

[0065] The term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Cationic lipids, including

[0066] The term "lipid nanoparticle" or "LNP" refers to a particle having at least one nanometer (nm) scale size (e.g., 1 to 1000 nm) containing one or more types of lipid molecules. The LNPs described herein may further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule partially or completely encapsulated within a lipid shell. Specifically, in some embodiments, the payload is a negatively charged molecule (e.g., mRNA encoding a therapeutic protein), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is anticipated that cationic lipids can interact with the negatively charged payload molecule and promote the incorporation and / or encapsulation of the payload into the LNP during LNP formation. Other lipids that can form a portion of the LNPs described herein include, but are not limited to, neutral and charged lipids, such as steroids, polymer-bound lipids, and various zwitterionic lipids.

[0067] The term "cationic lipid" refers to a lipid that carries a positive charge at any pH or hydrogen ion activity in its environment, or is capable of carrying a positive charge in response to the pH or hydrogen ion activity of its environment (e.g., its intended environment of use). Therefore, the term "cationic" encompasses both "permanent cation" and "cationizable." In some embodiments, the positive charge in the cationic lipid is caused by the presence of a quaternary nitrogen atom. In some embodiments, the cationic lipid comprises a zwitterionic lipid that carries a positive charge in its intended environment of use (e.g., at physiological pH). The cationic lipid can be one of the following: MC3, DOTAP, HL1001, ALC-0315, SM102, Dlin-MC3-DMA, DODMA, C12-200, DlinDMA, etc., with the following structures:

[0068] It is worth noting that MC3, DOTAP, HL1001, ALC-0315, SM102, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA are merely examples of cationic lipids. Any structure in the prior art that can be used to achieve delivery of LNP combination formulations is within the scope of protection of this invention.

[0069] Invention Details

[0070] The first aspect of this invention relates to a recombinant molecule

[0071] Recombinant protein

[0072] A recombinant protein includes three elements: a signal peptide, a GLP-1 receptor agonist, and a self-cleaving peptide. Preferably, these elements are sequentially linked from 5' to 3', i.e., signal peptide-GLP-1 receptor agonist-self-cleaving peptide. Preferably, the recombinant protein may further be:

[0073] Signal peptide - GLP-1 receptor agonist - self-cleaving peptide - signal peptide - GLP-1 receptor agonist... (and so on)

[0074] The length does not need to be specifically limited, but if so, the following structure is preferred:

[0075] Signal peptide-GLP-1 receptor agonist-autosplicing peptide-signal peptide-GLP-1 receptor agonist-autosplicing peptide-signal peptide-GLP-1 receptor agonist

[0076] The signal peptide is not particularly limited, as long as it is used for guidance and localization, and the post-translational excision is for the purpose of forming a fully functional protein, such as the proinsulin signal peptide. This is just an example, such as MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO:18), but it is not limited to this signal peptide. Any signal peptide that can achieve guidance and localization and whose post-translational excision does not affect activity is within the scope of protection.

[0077] Self-cleaving peptides are not particularly limited; any element capable of self-cleaving is acceptable. For example, it could be a P2A peptide, as shown in GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:20), or a T2A peptide, as shown in GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:21), or an E2A peptide, as shown in GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:22).

[0078] The GLP-1 receptor agonist is selected from liraglutide, exenatide, telpoxetine, dulaglutide, abiglutide, tasglutide, lixinatide, semaglutide, or any combination thereof. Specifically, the GLP-1 receptor agonist can be a single peptide or a fusion peptide of multiple peptides, preferably a fusion of 2-3 peptides, which may or may not be linked by a linker peptide, but are preferably linked by a linker peptide. The GLP-1 receptor agonist is not limited to the above selections.

[0079] Furthermore, it can also be selected from various receptor agonists such as GLP-1R / GIPR / GCGR.

[0080] Specifically, the GLP-1 receptor agonist is exemplary selected from...

[0081] Exendin-4:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO:17)

[0082] Exendin-3:HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO:19)

[0083] liraglutide:HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG(SEQ ID NO:23)

[0084] GLP-1 sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 24)

[0085] PB105 sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPC (SEQ ID NO: 25)

[0086] The first 31 amino acids (TR) at the N-terminus of Dulaglutide: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO:26)

[0087] The sequence comprises at least one of the following: a sequence having at least 90% identity to achieve the same function; and a sequence in which one or more amino acid residues are substituted, added, inserted, or deleted, preferably 1-3 residues; which is also the active sequence produced after translation.

[0088] The sequence of the recombinant protein, for example, can be any one of SEQ ID NO:3, 16, 17, 19, 23-26, or a sequence with more than 90% identity to achieve the same function, and can be a sequence in which one or more amino acid residues are substituted, added, inserted, or deleted, preferably 1-3 residues.

[0089] In one specific implementation, the recombinant protein sequence can be:

[0090] Nucleic acid molecules

[0091] Nucleic acid molecules capable of expressing the recombinant protein described above include DNA molecules and RNA molecules. Exemplarily, DNA molecules can be SEQ ID NO:1, 4, 6, 8, 10, 12, 14, or sequences with over 90% identity in achieving the same function, and sequences in which one or more amino acid residues are substituted, added, inserted, or deleted, preferably 1-3 residues. However, these are merely examples; in practice, depending on codon preferences, any DNA molecule sequence capable of expressing the recombinant protein described above is within the scope of protection. Exemplarily, RNA molecules can be SEQ ID NO:2, 5, 7, 9, 11, 13, 15, or sequences with over 90% identity in achieving the same function, and sequences in which one or more amino acid residues are substituted, added, inserted, or deleted.

[0092] In one specific implementation, the DNA molecular sequence of the recombinant protein sequence can be one of the following sequences.

[0093] CWT ORF DNA

[0094] CR026-01 ORF DNA

[0095] CR058-01 ORF DNA

[0096] CR053-FL14 ORF DNA

[0097] CR053-FL24 ORF DNA

[0098] CR053-FL85 ORF DNA

[0099] CR053-FPE ORF DNA

[0100] In one specific implementation, the RNA molecule sequence of the recombinant protein sequence can be one of the following sequences.

[0101] CWT ORF RNA

[0102] CR026-01 ORF RNA

[0103] CR058-01 ORF RNA

[0104] CR053-FL14 ORF RNA

[0105] CR053-FL24 ORF RNA

[0106] CR053-FL85 ORF RNA

[0107] CR053-FPE ORF RNA

[0108] The second aspect of this invention specifically relates to expression vectors

[0109] An expression vector capable of expressing the above-mentioned recombinant protein;

[0110] An expression vector comprising the above-mentioned recombinant nucleic acid molecules;

[0111] An expression vector comprising a nucleic acid sequence capable of expressing a glucagon-like peptide-1 (GLP-1) receptor agonist protein, wherein the nucleic acid sequence may be a DNA or RNA sequence; the glucagon-like peptide-1 (GLP-1) receptor agonist protein may be the recombinant protein described in the first aspect of the present invention;

[0112] The expression vector can be a DNA molecule, plasmid, linear precursor RNA, circular RNA molecule, etc.; circular RNA molecules are preferred.

[0113] In one specific implementation, the expression vector can be a linear precursor RNA.

[0114] Linear precursor RNA can circularize into circular RNA molecules, including homologous arms, introns, spacers, internal ribosome entry sites, and coding regions;

[0115] Further, it may also include IG5 and IG3 sequences, namely the 6-11nt region of introns in the P1 stem-loop domain;

[0116] Furthermore, E1 and E2 (exon) sequences can improve circularization efficiency and increase the success rate of circularization.

[0117] IG5 and IG3 form a complementary pair with E2 in terms of structure, which helps to stabilize the substrate structure. IG5 and IG3 can change accordingly with changes in the exon sequence.

[0118] The coding region can be an active sequence, such as the aforementioned recombinant RNA molecule, for example, the sequence shown in SEQ ID NO:2, 5, 7, 9, 11, 13, 15;

[0119] Specifically, the following constructor is an example (see Figure 1 for the structure diagram):

[0120] Constructor-FL

[0121] 5' outer homologous arm - IG5 - 5' intron - 3' intron - E2 - 5' spacer - Internal ribosome entry site (IRES) - coding region - 3' spacer - E1 - IG3 - 3' outer homologous arm

[0122] Constructor - AZ

[0123] 5' outer homologous arm - 3' intron - 5' spacer region - internal ribosome entry site (IRES) - coding region - 3' spacer region - 5' intron - 3' outer homologous arm

[0124] DNA molecules

[0125] The present invention further relates to DNA molecules capable of preparing the above-mentioned linear RNA precursor molecules, wherein the coding region may be SEQ ID NO:1, 4, 6, 8, 10, 12, 14.

[0126] In one specific implementation, the RNA sequence of the construct-FL element may be as follows:

[0127] 5' outer homologous arm - FL RNA sequence: GGGAGACCCUCGAAUGGAAAUU (SEQ ID NO:27)

[0128] IG5-FL RNA sequence: CUGU (SEQ ID NO:28)

[0129] 5' intron-FL RNA sequence:

[0130] 3' intron-FL RNA sequence:

[0131] E2-FL RNA sequence: AACA (SEQ ID NO:31)

[0132] 5' spacer region - FL RNA sequence: AAACAAAGCGAAUAGCCGAGAUC (SEQ ID NO:32),

[0133] IRES-OR6-FL RNA sequence:

[0134] 3' spacer region - FL RNA sequence: GUACCGGCUAUUCGCAAACAA (SEQ ID NO:34)

[0135] E1-FL RNA sequence: GAU (SEQ ID NO:35)

[0136] IG3-FL RNA sequence: AACA (SEQ ID NO:36)

[0137] 3' outer homologous arm - FL RNA sequence: AAAUUUCCAUUUAUCAGAUCUAGA (SEQ ID NO:37)

[0138] In one specific implementation, the DNA sequence of the construct-FL element can be as follows: 5' outer homologous arm-FL DNA sequence: GGGAGACCCTCGAATGGAAATT (SEQ ID NO:38) IG5-FL DNA sequence: CTGT (SEQ ID NO:39)

[0139] 5' intron-FL DNA sequence:

[0140] 3' intron-FL DNA sequence:

[0141] E2-FL DNA sequence: AACA (SEQ ID NO:42)

[0142] 5' spacer region - FL DNA sequence: AAACAAAGCGAATAGCCGAGATC (SEQ ID NO:43),

[0143] Internal ribosome entry site (IRES)-OR6-FL DNA sequence:

[0144] 3' spacer region - FL DNA sequence: GTACCGGCTATTCGCAAACAA (SEQ ID NO:45) E1-FL DNA sequence: GAT (SEQ ID NO:46)

[0145] IG3-FL DNA sequence: AACA (SEQ ID NO:47)

[0146] 3' External homologous arm - FL DNA sequence: AAATTTCCATTTATCAGATCTAGA (SEQ ID NO:48) In one specific embodiment, the RNA sequence of the construct-AZ element can be as follows: 5' External homologous arm - AZ RNA sequence: GGGAGACCCUCGAAUGGAAUU (SEQ ID NO:49) 3' Intron - AZ RNA sequence:

[0147] 5' spacer region - AZ RNA sequence:

[0148] IRES-CVB3-AZ RNA sequence:

[0149] 3' spacer region - AZ RNA sequence: AAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGA (SEQ ID NO:53)

[0150] 5' intron-AZ RNA sequence:

[0151] 3' External homologous arm - AZ RNA sequence: AAUUCCAUUUAUCAGAUUUCUAGA (SEQ ID NO:55)

[0152] In one specific implementation, the DNA sequence of the construct-AZ element may be as follows:

[0153] 5' External Homologous Arm - AZ DNA Sequence: GGGAGACCCTCGAATGGAATT (SEQ ID NO:56)

[0154] 3' intron-AZ DNA sequence:

[0155] 5' spacer region - AZ DNA sequence:

[0156] IRES-CVB3-AZ DNA sequence:

[0157] 3' spacer region - AZ DNA sequence: AAAAAACAAAAAACAAAACGGCTATTATGCGTTACCGGCGA (SEQ ID NO:60)

[0158] 5' intron-AZ DNA sequence:

[0159] 3' External homologous arm - AZ DNA sequence: AATTCCATTTATCAGATTTCTAGA (SEQ ID NO:62)

[0160] Specific examples are shown in Tables 1 and 2.

[0161] Table 1. Linear precursor RNA molecular elements: RNA sequence and DNA sequence (construct FL)

[0162] Table 2. Linear precursor RNA molecular elements: RNA and DNA sequences (construct-AZ)

[0163] In one specific embodiment, the sequences shown in SEQ ID NO:2, 5, 7, 9, 11, 13, and 15 can be introduced into the linear precursor RNA molecule as coding region RNA sequences. Although it is not necessary to limit which circularization system to be used, it is preferable to embed the coding regions shown in SEQ ID NO:2 and 5 into the ORF region of the construct-AZ, and it is preferable to embed the coding regions shown in SEQ ID NO:7, 9, 11, 13, and 15 into the ORF region of the construct-AFL.

[0164] In one specific embodiment, a linear precursor RNA molecule as shown in SEQ ID NO: 63-69 is further provided.

[0165] AZ construct linear precursor RNA of sequence 63 CWT

[0166] AZ construct linear precursor RNA of sequence 64 CR026-01

[0167] FL construct linear precursor RNA of sequence 65 CR058-01

[0168] FL construct linear precursor RNA of sequence 66 CR053-FL14

[0169] FL construct linear precursor RNA of sequence 67 CR053-FL24

[0170] FL construct linear precursor RNA of sequence 68 CR053-FL85

[0171] FL construct linear precursor RNA of sequence 69 CR053-FPE

[0172] In one specific embodiment, a template DNA molecule, as shown in SEQ ID NO: 70-76, for synthesizing circular RNA is further provided.

[0173] CWT's AZ construct is used to synthesize template DNA molecules for circular RNA.

[0174] The AZ construct of CR026-01 is used to synthesize template DNA molecules for circular RNA.

[0175] The FL construct of CR058-01 is used to synthesize template DNA molecules for circular RNA.

[0176] The FL construct of CR053-FL14 is used to synthesize template DNA molecules for circular RNA.

[0177] The FL construct of CR053-FL24 is used to synthesize template DNA molecules for circular RNA.

[0178] AGA (SEQ ID NO:74)

[0179] The FL construct of CR053-FL85 is used to synthesize template DNA molecules for circular RNA.

[0180] The FL construct of CR053-FPE is used to synthesize template DNA molecules for circular RNA.

[0181] In one specific embodiment, the present invention relates to circular RNA molecules.

[0182] The circular RNA molecule is formed by circularizing the aforementioned linear precursor RNA molecule.

[0183] In one specific embodiment, the present invention provides multiple circular RNA molecules (wherein the circular RNA structure formed by circularization of construct AZ is shown in Figure 2, and the circular RNA structure formed by circularization of construct FL is shown in Figure 3), further wherein CWT and CR026-01 are formed by circularization of construct AZ, and further wherein CR058-01, CR053-FL14, CR053-FL24, CR053-FL85, and CR053-FPE are formed by circularization of construct FL. The sequences shown below are circular RNAs connected end-to-end. For example, they may be as shown in SEQ ID NO: 77-83.

[0184] CWT circular RNA sequence

[0185] CR026-01 circular RNA sequence

[0186] CR058-01 circular RNA sequence

[0187] CR053-FL14 circular RNA sequence

[0188] CR053-FL24 circular RNA sequence

[0189] CR053-FL85 circular RNA sequence

[0190] CR053-FPE circular RNA sequence

[0191] A host cell containing the above-mentioned expression vector, which can be a prokaryotic or eukaryotic cell, as long as it can be used for the expression of biological macromolecule proteins, preferably a eukaryotic cell; including but not limited to HEK293T, HEK293, BHK, CHO, 3T3, NSO, HeLa, HT-1080, PERC6, CAP, HKB-11, Huh-7, etc.

[0192] The third aspect of this invention relates to a method for preparing an expression vector.

[0193] Design linear precursor RNA molecular sequences, including DNA sequences and RNA sequences.

[0194] After plasmid extraction, linearized templates are prepared by restriction endonuclease digestion to obtain linear precursor RNA, such as linear precursor RNA LWT, LR026-01, LR058-01, LR053-FL14, LR053-FL24, LR053-FL85 and LR053-FPE (RNA sequences shown in SEQ ID NO:63-69).

[0195] circRNA was synthesized using a circularization system comprising a linear precursor RNA molecule, ATP / CTP / UTP / GTP, RNA polymerase, inorganic pyrophosphatase, and the linear precursor RNA molecule template. After processing, the circRNA was digested with RNase R to purify the circular RNA.

[0196] The final result is a circular RNA molecule, for example, a plasmid with a promoter and construct designed and synthesized by SEQ, to obtain a plasmid strain with the target sequence, wherein the target sequence is the circular RNA sequence shown in SEQ ID NO:77-83, which is the DNA sequence of the linear precursor RNA.

[0197] The fourth aspect of the present invention relates to a composition

[0198] Furthermore, the present invention also provides compositions that can be pharmaceutical compositions, or more specifically, innovative drug delivery and expression platforms, including but not limited to delivery systems such as LNPs, liposomes, and exosomes for delivering the aforementioned RNA vectors, such as circular RNA molecules.

[0199] Preferably, the present invention promotes the use of an LNP delivery system, wherein the LNP composition comprises the following components:

[0200] (1) Cationic lipids: The cationic lipids are selected from SM-102, ALC-0315, HL1001, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA; preferably SM-102 and HL1001.

[0201] (2) The auxiliary lipid (phospholipid) is selected from DSPC, DOPE, DOPC, DOPG or DOPS; preferably, the auxiliary lipid is DOPE.

[0202] (3) Structural lipids: cholesterol or cholesterol derivatives

[0203] (4) PEG-lipids

[0204] The PEG-lipid can be mPEG2000-DMG, DSG-PEG2000, ALC-0519, etc.

[0205] Furthermore, the present invention provides an LNP composition.

[0206] The molar ratio of SM102 / HL1001: cholesterol: DSPC: mPEG-DMG-2K is (25-65): (25-70): (5-25): (0.5-5), and the concentration of the lipid solution can be between 5 mg / mL and 15 mg / mL.

[0207] More preferably, the molar ratio can be (23-61):(27-66):(8-22):(0.7-4), even more preferably, it can be (27-58):(30-60):(8-19):(0.7-3.5), and even more preferably, it can be (30-55):(30-50):(8-16):(0.7-2.0).

[0208] The concentration of the lipid solution can be 5-10 mg / mL, more preferably 5 mg / mL, 7 mg / mL, 10 mg / mL, 12 mg / mL, or 15 mg / mL, and even more preferably selected from 7 mg / mL, 10 mg / mL, or 12 mg / mL.

[0209] Route of administration: The composition formulations provided by this invention can be used in the composition by injection at various sites such as intravenous, intramuscular, subcutaneous, intradermal, and intraperitoneal administration, depending on many individual factors such as age, disease severity, total weight, sex, and other alleviating factors.

[0210] Dosing frequency: The dosing frequency does not need to be specifically limited and needs to be based on various individual factors. However, in general, compared with GLP-1 inhibitor-related protein injection alone, it can reduce the dosing frequency and improve medication compliance. The dosing frequency can range from once a week to once a year. Specifically, it can be once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every six months, or once a year. The preferred frequencies are once a week, once every two weeks, once a month, and once every three months.

[0211] The fifth aspect of this invention relates to uses

[0212] The aforementioned recombinant proteins, recombinant nucleic acid molecules, expression vectors, host cells, and pharmaceutical compositions are used in the treatment and prevention of GLP-1-related metabolic diseases, and in the preparation of medicaments for the treatment of GLP-1-related metabolic diseases.

[0213] The aforementioned uses of recombinant proteins, recombinant nucleic acid molecules, expression vectors, host cells, and compositions in assisting weight loss, as well as their use in preparing foods and health products with weight loss functions.

[0214] Metabolic diseases

[0215] The metabolic diseases or conditions mentioned include diabetes, obesity, dyslipidemia, elevated glucose levels, elevated insulin levels, and diabetic nephropathy.

[0216] Hyperglycemia is normalized through glucose-dependent, insulin-dependent, and insulin-independent mechanisms.

[0217] Hyperglycemia related: The use of the compositions involved in this invention in methods of treating diabetes and preventing hyperglycemia, and in the preparation of medicaments for treating diabetes and preventing hyperglycemia;

[0218] The diabetes mellitus referred to includes type 1 or type 2 diabetes mellitus, early-onset type 2 diabetes mellitus, idiopathic type 1 diabetes mellitus (type 1b), juvenile-onset atypical diabetes mellitus (YOAD), adult-onset diabetes mellitus in adolescents (MODY), latent autoimmune diabetes mellitus in adults (LADA), malnutrition-related diabetes mellitus, gestational diabetes mellitus, diabetic nephropathy, impaired glucose tolerance, diabetic neuropathy, and diabetic retinopathy.

[0219] Hyperinsulinemia, impaired glucose metabolism, insulin resistance, hepatic insulin resistance

[0220] A method for regulating insulin levels in patients

[0221] A method for regulating glucose levels in a patient includes lowering fasting plasma glucose levels.

[0222] Weight loss related: The compositions of this invention are used in the preparation of foods or pharmaceuticals for individuals in need of controlling weight, preventing weight gain and promoting weight loss, reducing fat, controlling appetite, and treating all types of obesity, including morbid obesity. The food compositions may be functional foods. Furthermore, they are used in weight control for the further stabilization of blood glucose, cholesterol, and triglyceride levels.

[0223] Obesity: Use of the compositions of the present invention in the preparation of medicaments for treating conditions caused by or characterized by overweight.

[0224] To verify the above-mentioned uses, the present invention further verified the expression of the prepared circular RNA molecules at the cellular level by transfecting circRNA samples into mammalian cells and detecting the expression levels of exogenous proteins at different time ranges, such as the expression levels of exogenous proteins, such as Exendin-4 related proteins, at 24h, 48h, 72h, and 168h.

[0225] The LNP formulation was prepared and its physicochemical properties were comprehensively tested, including nucleic acid content, encapsulation efficiency, nanoparticle size, dispersion index (PI), zeta potential, microbial limit, and endotoxin content, to verify its homogeneity and stability.

[0226] To verify functional activity, cells overexpressing the GLP-1 receptor can be used for functional testing, including but not limited to CHOK1, CHO AM1D, HEK293T, U2OS, INS1 832 / 3, HEK293 cells, HeLa cells, and BHK cells. cAMP accumulation can be measured to determine the activity of circular RNA molecules.

[0227] To verify functional activity, formulations containing circular RNA molecules can be used in model organisms for experiments, preferably mammals, including but not limited to rats, mice, rabbits, cats, dogs, humans, and monkeys. Of course, many specific types are also included, such as BalB / C mice, C57 mice, rhesus monkeys, cynomolgus monkeys, beagles, rats, miniature pigs, etc. (to name only). Experiments include a variety of animal models that can be established to detect the use of circular RNA molecule formulations in weight loss, weight control, blood glucose control, blood glucose reduction, treatment of obesity, treatment of diabetes, prevention of diabetes, and control of GLP-1-related metabolic diseases.

[0228] Specifically, it can be used to control weight loss in diet-induced obesity, reduce body fat, and control related blood routine indicators in animal models, such as total cholesterol, high-density lipoprotein, low-density lipoprotein, blood lipids, blood glucose, and triglycerides. Beneficial effects

[0229] This invention overcomes the limitations of existing technologies by designing and optimizing a circular RNA sequence that encodes and expresses a GLP-1 receptor agonist. Due to its unique circular structure, the circular RNA exhibits higher stability and resistance to degradation, significantly extending the half-life of the GLP-1 receptor agonist and reducing the frequency of administration. Compared to conventional linear mRNA, the circular RNA demonstrates higher translation efficiency and avoids the protein activity loss that may occur with conventional modification methods.

[0230] Furthermore, this invention further improves the circularization efficiency of circular RNA and the protein expression level after transfection by optimizing codons and the secondary structure of circular RNA. Codon optimization improves translation efficiency and enhances the expression of the target protein in target cells. Simultaneously, by optimizing the design of the circular RNA secondary structure, it is made easier to achieve efficient circularization, thereby increasing its stability and ribosome reloading capacity, and ultimately improving the yield of protein translation.

[0231] Therefore, the circular RNA of this invention not only efficiently translates GLP-1 receptor agonist proteins, but also further improves its expression efficiency through codon and structural optimization, ensuring long-term efficacy and making it suitable for long-term glycemic control and weight management. This optimization scheme results in higher protein expression levels of the circular RNA after cell transfection, demonstrating superior efficacy compared to existing protein drugs, reducing the burden of frequent dosing, and improving patient compliance and quality of life. This invention provides an innovative drug delivery and expression platform suitable for treating diabetes, obesity, and other metabolic diseases. Attached Figure Description

[0232] Figure 1 is a schematic diagram of the linear precursor RNA sequence design in this invention;

[0233] Figure 2 is a schematic diagram of the circular RNA sequence after the construct-AZ is circularized;

[0234] Figure 3 is a schematic diagram of the circular RNA sequence after the construct-FL is circularized. Detailed Implementation

[0235] Example 1: Preparation of circular RNA molecules

[0236] 1.1 Sequence design for circular linear precursor RNA

[0237] This invention designed and synthesized the following optimized nucleic acid sequences encoding exenatide-related peptides, using the unoptimized exenatide-encoding nucleic acid sequence CWT as a control. The nucleic acid sequences encoding exenatide-related peptides (CWT, CR026-01, CR058-01, CR053-FL14, CR053-FL24, CR053-FL85, and CR053-FPE) are shown in Table 3.

[0238] Table 3: Sequences of CR026-01, CR058-01, CR053-FL14, CR053-FL24, CR053-FL85, and CR053-FPE

[0239] (The numbers in the table are the serial numbers of SEQ ID NO)

[0240] When designing circular RNAs, multiple factors need to be considered to optimize their function and expression efficiency. First, the CDS sequence of the target protein must be optimized according to the codon preference of the host cell to improve translation efficiency. Second, the secondary structure of the circular RNA needs to be optimized to enhance its stability. GC% content is also a key influencing factor, directly affecting the structure and stability of the RNA. Furthermore, the interaction between the CDS sequence and IRES elements must be minimized to ensure efficient translation. Therefore, this design comprehensively optimizes the process by combining codon preference, the stability of the sequence secondary structure, GC% content, and the interaction between the CDS and IRES elements. The codon fitness index (CAI), minimum free energy (MFE), GC% content, and assembly diversity of each nucleic acid sequence are shown in Table 4.

[0241] Table 4 shows the codon fitness index (CAI) and minimum free energy (MFE) of the nucleic acid sequences encoding exenatide-related peptides.

[0242] (AzoPIE is called the AZ construct, and FlexCirc is called the -FL construct)

[0243] The molecular weight exponents (MFE) of CR026-01 and CR058-01 is -121.2 kcal / mol, indicating high structural stability. However, the MFE of CR053-FPE is -476 kcal / mol, significantly lower than other sequences, suggesting even greater structural stability. Lower MFE generally indicates greater structural stability and a higher probability of folding. The ultra-low MFE of CR053-FPE may indicate higher cyclization efficiency and greater resistance to degradation.

[0244] The CAI of CR053-FL85 was the highest at 0.971, indicating that the codons of this sequence are closest to the highly expressed gene, and the translation efficiency may be the highest. Overall, the CAI values ​​were all high (close to 1), which means that the optimized sequences all have good translation efficiency.

[0245] GC content has a significant impact on RNA structural stability. The GC% of CR053-FL85 is 0.702, slightly higher than other sequences; high GC content generally contributes to structural stability. Overall, the GC% varies little, with all sequences falling between 0.67 and 0.70, suggesting their structures are likely relatively stable.

[0246] CR053-FL85 and CR053-FL24 have the lowest ensemble diversity values, at 130.73 and 131.54 respectively. This indicates that they have minimal interference with IRES, relatively simple structures, and high predicted translation efficiency and expression. In contrast, CR026-01 and CR058-01 have an ensemble diversity of 177.09, suggesting higher IRES ensemble diversity, which may result in slightly greater interference with IRES and potentially lower translation efficiency.

[0247] Based on the above sequences, the linear template DNA (LWT, LR026-01, LR058-01, LR053-FL14, LR053-FL24, LR053-FL85, and LR053-FPE) used in this embodiment for circularization, wherein LR058-01, LR053-FL14, LR053-FL24, LR053-FL85, and LR053-FPE contain 5' segments from the 5' end to the 3' end. The outer homologous arm, IG5, 5' intron, 3' intron, E2, 5' spacer region, internal ribosome entry site (IRES), coding region, 3' spacer region, E1, IG3, and 3' outer homologous arm; LWT, LR026-01 contains the following components from the 5' end to the 3' end: 5' outer homologous arm, 3' intron, 5' spacer region, internal ribosome entry site (IRES), coding region, 3' spacer region, 5' intron, and 3' outer homologous arm;

[0248] Constructor-FL

[0249] 5' outer homologous arm - IG5 - 5' intron - 3' intron - E2 - 5' spacer - internal ribosome entry site (IRES) - coding region - 3' spacer - E1 - IG3 - 3' outer homologous arm

[0250] Constructor - AZ

[0251] 5' external homologous arm - 3' intron - 5' spacer region - IRES - coding region - 3' spacer region - 5' intron - 3' external homologous arm

[0252] LR058-01, LR053-FL14, LR053-FL24, LR053-FL85, and LR053-FPE use the FL construct;

[0253] LWT and LR026-01 use the AZ construct.

[0254] The linear precursor RNA molecules shown in SEQ ID NO:63-69 were constructed.

[0255] 1.2 Plasmid Preparation

[0256] A pUC57 plasmid (containing SEQ ID NO: 70-76) containing the T7 promoter sequence (TAATACGACTCACTATAGGG) and the DNA sequence of the above-mentioned construct was designed and the whole genome was synthesized (Suzhou Genewiz Biotechnology Co., Ltd.) to obtain a pUC57 plasmid strain containing the target sequence.

[0257] Bacterial amplification

[0258] The pUC57 plasmid strain containing the target sequence, provided by a third-party sequence synthesis company, was extracted from the original glycerol strain frozen at -80℃ and inoculated into a shake flask at a ratio of 0.2%, and cultured overnight at 37℃ and 220 rpm on a shaker.

[0259] plasmid extraction

[0260] Plasmids were extracted according to the instructions of the QIAGEN endotoxin-free plasmid extraction kit (catalog number: 12362), and the content and purity of the obtained plasmids were tested.

[0261] 1.3 Linearization Template Preparation

[0262] The synthesized plasmid was linearized and digested using EcoRI restriction endonuclease to prepare an IVT transcription template. The digestion system is shown in Table 5.

[0263] Table 5. Formula for EcoR I enzyme digestion system (500 μL)

[0264] Prepare 400 μL reaction systems according to Table 5, place the reaction systems in a metal water bath, and react at 37°C for 3 hours. After the reaction is complete, terminate the enzymatic digestion reaction at 65°C for 20 minutes. Linearized template was recovered using the Wizard SV Gel and PCR clean-up system purification kit. An equal amount of membrane binding buffer was added to each digestion system and mixed thoroughly. SV columns were inserted into collection tubes, and the mixture was transferred to the SV columns and incubated at room temperature for 1 min. The SV column assembly was then centrifuged at 14000 rpm for 1 min. The SV columns were removed, and the liquid in the collection tube was discarded. 700 μL of membrane washing buffer was added to the SV columns, and the mixture was centrifuged at 14000 rpm for 1 min. The liquid in the collection tube was discarded. Then, 500 μL of membrane washing buffer was added to the SV columns, and the mixture was centrifuged at 14000 rpm for 5 min. The liquid in the collection tube was discarded. Finally, the SV column assembly was centrifuged again for 1 min to evaporate the ethanol. The SV columns were then transferred to 1.5 ml centrifuge tubes, and 100 μL of nuclease-free water was added. The mixture was incubated at room temperature for 1 min, centrifuged at 14000 rpm for 1 min, and the SV columns were discarded. The eluted sample was kept as the collected linearized template. Samples from parallel groups were combined into one sample. The linearized templates of each sequence were sampled separately and their purity was detected by HPLC to ensure that the prepared linearized templates met the requirements for subsequent IVT transcription.

[0265] The linear template DNAs LWT, LR026-01, LR058-01, LR053-FL14, LR053-FL24, LR053-FL85 and LR053-FPE shown in SEQ ID NO:70-76 were finally obtained.

[0266] 1.4 Preparation of circular RNA

[0267] Synthesize circRNA samples using the linear template DNA according to the system shown in the table below.

[0268] Table 6 IVT in vitro transcription system

[0269] The linear template DNA was used to prepare the in vitro transcription reaction system according to the table above. The transcription system was placed in a benchtop insulated shaker and reacted at 45°C for 2 hours. After the reaction was completed, 75 μL of DNase I and 175 μL of 10×DNase I buffer were added to the reaction system, and the reaction was carried out at 37°C for 15 minutes. 875 μL of RNA precipitation solution with 7.5M lithium chloride and 50 mM EDTA was added, and the mixture was mixed and precipitated at -20°C for 1 hour. After precipitation, the mixture was centrifuged at 18360×g for 15 minutes at 4°C to remove as much supernatant as possible. 1312.5 μL of 70% ethanol solution was added, and the precipitate was washed. The mixture was centrifuged again under the same conditions to remove as much supernatant as possible, and the ethanol was evaporated. The precipitate was resuspended in water for injection to obtain circRNA samples. RNA concentration and HPLC purity were measured separately, and the remaining samples were temporarily stored at -80°C.

[0270] circRNA sample RNase R digestion treatment

[0271] The circRNA samples were digested with RNase R enzyme. The RNase R enzyme digestion system is shown in Table 7.

[0272] Table 7 RNase R enzyme digestion system

[0273] circRNA samples were prepared using a 10000 μL RNase R digestion reaction system according to Table 7. The reaction system was placed in a benchtop constant temperature shaker and reacted at 37℃ for 30 min. After the reaction, 5000 μL of RNA precipitation solution (7.5 M lithium chloride and 50 mM EDTA) was added to each reaction system, mixed well, and precipitated at -20℃ for 1 h. After precipitation, the precipitate was centrifuged at 18360 × g for 15 min at 4℃, and the supernatant was removed as much as possible. 7500 μL of 70% ethanol solution was added, and the precipitate was washed by rinsing. The precipitate was centrifuged again under the same conditions to remove as much supernatant as possible, and the ethanol was evaporated to dryness. The precipitate was resuspended in 2500 μL of water for injection. Samples were sent for RNA concentration and HPLC purity determination.

[0274] Purification of circular RNA

[0275] The circRNA sample was purified using the SCG-P 100 protein purification system. The flow rate was 3 mL / min. After equilibration with 30% B solution for 5 CV, 3 mg of sample was loaded. The elution peak was collected, and the sample was concentrated by ultrafiltration and the buffer was changed. The sample purity reached over 90% after chromatography. The purified circular peak was collected, and the circular RNA was precipitated with LiCl to obtain the purified circular RNA shown in SEQ ID NO:77-83, which was used for subsequent experiments.

[0276] Example 2: Expression of circular RNA at the cellular level

[0277] Cell plating:

[0278] HEK293T cells with 90% confluence were collected, the cell culture medium was discarded, and the cells were washed with PBS. The PBS was discarded, and the cells were digested with 0.05% trypsin. After the cells became rounded, an appropriate amount of antibiotic-free cell culture medium (DMEM containing 10% FBS) was added, and the cells were pipetted several times. The cells were then transferred to a 50mL centrifuge tube, centrifuged at 800rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended with an appropriate amount of antibiotic-free cell culture medium. Cell counting was performed, and the cell concentration was adjusted to 1×106 cells / mL. 1mL of the cell culture medium was seeded into 12-well plates, and the cell culture plates were then incubated at 37℃ in a 5% CO2 incubator for 24h.

[0279] Transfection:

[0280] Table 8 RNA dosage

[0281] For each sample, solutions A and B were prepared according to Table 8, gently mixed, and allowed to stand at room temperature for 5 minutes each. Solution B was added to solution A, gently mixed, and allowed to stand at room temperature for 20 minutes. Three plates of cells were removed from the incubator the previous day, and 200 μL of the above transfection mixture was added dropwise to each plate of cells. After gentle mixing, the plates were incubated at 37°C in a 5% CO2 incubator. After 4 hours, the incubator was discarded, and the cells were replaced with 1 mL of DMEM containing 5% FBS and cultured for 24 h, 72 h, and 168 h, respectively.

[0282] Blank control: HEK293T cells without circRNA transfection were set as blank control at each time point.

[0283] Protein collection from supernatant:

[0284] At approximately 24h, 72h, and 168h after transfection, the cell culture plates were removed from the CO2 incubator, and the supernatant was collected into 1.5mL centrifuge tubes. The tubes were centrifuged at 1000g and 4℃ for 10 minutes to remove cell debris, and the supernatant was collected into new 1.5mL centrifuge tubes.

[0285] ELISA test reagent preparation

[0286] Exendin-4 ELISA Kit (Abcam)

[0287] Solution preparation: Preparation of standard stock solution (6000 pg / mL): Add Sample Diluent NS (the reconstitution volume is indicated on the label of the standard) to an Exendin-4 Lyophilized Recombinant Protein tube, keep at room temperature for 10 minutes and mix gently to obtain a 6000 pg / mL stock solution, dispense 0.1 mL / tube, and store at -80℃.

[0288] Prepare 8 EP tubes, labeled ST1-ST8. Add 350 μL of Sample Diluent NS to ST1, and 150 μL of Sample Diluent NS to ST2-ST8. Add 50 μL of standard stock solution (6000 pg / mL) to ST1 and mix well. Add 150 μL of ST1 to ST2 and mix well. Add 150 μL of ST2 to ST3 and mix well, and so on, diluting to ST7. ST8 contains no protein standard and serves as a blank control. The protein concentrations of ST1-ST8 are 750 pg / mL, 375 pg / mL, 187.5 pg / mL, 93.75 pg / mL, 46.88 pg / mL, 23.44 pg / mL, 11.72 pg / mL, and 0 pg / mL, respectively.

[0289] 1×Wash Buffer PT: Dilute 10×Wash Buffer PT with deionized water at a ratio of 10:1 before use.

[0290] Antibody Cocktail: Dilute 10×Exendin-4Capture Antibody and 10×Exendin-4Detector Antibody with Antibody Diluent 4BI.

[0291] ELISA testing

[0292] Remove the microplate and add 50 μL of sample or standard to each well. Add 50 μL of Antibody Cocktail to each well. Cover the microplate with a sealing film and incubate at room temperature on a shaker at 400 rpm for 1 hour. Aspirate the contents of the wells and wash each well with 350 μL of 1×Wash Buffer PT, for a total of 3 washes. After the last wash, remove any residual solution by inverting the microplate and vigorously tapping it on a clean paper towel. Add 100 μL of TMB Development Solution to each well and incubate in the dark on a shaker at 400 rpm for 10 minutes. (The optimal incubation time may vary between 5 and 20 minutes depending on laboratory conditions). Add 100 μL of Stop Solution to each well and shake the microplate for 1 minute to mix. Measure the OD value at 450 nm using a microplate reader.

[0293] Cell supernatants from samples taken at different time points were diluted by different factors before ELISA detection. The data are shown in Table 9. No Exendin-4 protein expression was detected in the blank control.

[0294] Table 9-1 Exendin-4 protein expression levels at 24h, 72h, and 168h after circular RNA transfection.

[0295] Table 9-2 Exendin-4 protein expression levels 72 h after cell transfection with circular RNA

[0296] Table 9-3 Exendin-4 protein expression levels 168 h after cell transfection with circular RNA

[0297] Table 9 shows that circRNAs can be expressed efficiently and continuously within 7 days of transfecting cells. The CR026-01 sample expressed Exendin-4 protein, with expression levels of 34557.4 pg / mL, 71278.5 pg / mL, and 91003.8 pg / mL at 24 h, 72 h, and 168 h, respectively. CR058-01, CR053-FL14, CR053-FL24, CR053-FL85, and CR053-FPE all use FlexCirc as their circular backbone. Except for CR063-FPE, the expression levels of the other molecular targets were found to be low, possibly because FlexCirc is not suitable for short proteins, and the circular RNA has low binding efficiency to ribosomes. The expression level was further increased by applying OR6 IRES due to the extension of the CR063-FPE sequence. The expression levels at 24h, 72h and 168h were 2.9 times, 3.1 times and 3.5 times that of CR026-01, respectively.

[0298] Example 3: Preparation of LNP formulation for delivering circular RNA

[0299] To successfully deliver circular mRNA into animals, this embodiment employs LNP delivery. Negatively charged circular RNA (circRNA) is microfluidically mixed with a mixture of lipid molecules dissolved in the organic phase (core ionizable lipid molecule SM-102, auxiliary phospholipid DOPE, PEGylated lipid molecule PEG-DMG2K, and cholesterol) to form homogeneous circRNA-LNP nanoparticles.

[0300] 3.1 Experimental Materials

[0301] The ionizable lipid molecule SM-102 phospholipid (DSPC) was purchased from Corden Pharma; cholesterol was purchased from Sigma-Aldrich; and mPEG2000-DMG (i.e., DMG-PEG 2000 or PEG-DMG2K) was purchased from Avanti Polar Lipids, Inc.

[0302] Table 10 Solution Preparation

[0303] NA indicates no detection

[0304] 3.2 Preparation of circular RNA-LNP encapsulated samples

[0305] circRNA dilution: Dilute CR026-01 Mix with an aqueous phase of Water240402 to an N / P ratio of 6.

[0306] Preparation of lipid solution: Dissolve SM102:Cholesterol:DSPC:mPEG-DMG-2K in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5 to prepare a lipid solution of 10 mg / mL.

[0307] Instrumentation: Microfluidic device. 10 mL of DR-CR026-01 was taken with a 10 mL syringe, and 3.4 mL of ethanol was taken with a 5 mL syringe. The total volume was set to 13.3 mL, the total flow rate to 20 mL / min, the ratio of aqueous to ethanol flow rates to 3:1, the initial waste liquid to 0.6 mL, and the final waste liquid to 0.2 mL. The sample was encapsulated in 7 separate batches and named CR026-01-HUO.

[0308] Dilution: Immediately after encapsulation, the sample CR026-01-HUO was diluted 5 times with CC240402 and named CR026-01-HUO-Pre TFF.

[0309] Liquid exchange and concentration: CR026-01-HUO-Pre TFF was first concentrated using TFF membrane ultrafiltration. The first step involved replacing the medium twice with CC240402, resulting in a 2-fold concentration. The second step involved replacing the medium four times with TBS240402, concentrating to approximately 50 mL. Afterwards, ultrafiltration was performed using an ultrafiltration tube for further concentration. The collected sample was named CR026-01-HUO-TFF end.

[0310] Sterilization collection: The actual amount of concentrated sample CR026-01-HUO-TUF end collected was 23.25 mL. 5.81 mL of cryoprotectant was added in the clean bench, and the sample was sterilized by passing it through a 0.22 μm filter membrane and named CR026-01-DP.

[0311] In this invention, other circular RNAs are also prepared using LNP delivery technology to produce circular RNA formulations, which are named CWT-DP, CR058-01-DP, CR053-FL14-DP, CR053-FL24-DP, CR053-FL8-DP, and CR053-FPE-DP, respectively.

[0312] 3.3 Detection of physicochemical properties of circular RNA-LNP formulations

[0313] Table 11. Detection results of physicochemical properties of the circular RNA-LNP preparation (CR026-01)

[0314] The experimental results are shown in Table 10. After encapsulation with LNP, the circular RNA formulation showed high encapsulation efficiency, and the nanoparticle size, dispersion coefficient, zeta potential and other indicators all showed good uniformity and stability.

[0315] NA indicates no detection

[0316] Example 4: Functional Activity of Circular RNA Molecules

[0317] The functional activity of the circular RNA preparation prepared in Example 3 was determined in the HEK-293T clonal cell line expressing GLP-1R.

[0318] Collection of supernatant from HEK-293T cells transfected with circular RNA: On day 1, cells were seeded in 24-well plates at a density of 2 × 10^5 cells / mL, with 500 μL of cell suspension per well. On day 2, circular RNA transfection was performed. Two mixtures were prepared: 25 μL of Opti-MEM medium and 1.5 μL of RNAiMAX transfection reagent (referred to as A), and 25 μL of Opti-MEM medium and 2 μg of circular RNA (referred to as B). A and B were thoroughly mixed and allowed to stand for 10–20 minutes before being added to the cells. Subsequently, on days 3–7 post-transfection, the supernatant was collected at 24, 72, and 168 hours, respectively. After each collection, the cells were centrifuged to remove cell debris, and the supernatant was stored at -80°C for subsequent cAMP assays.

[0319] cAMP assay of Exendin-4 peptide in cell supernatant: using cAMP accumulation was measured using the Ultra cAMP kit (PerkinElmer). HEK-293T clones overexpressing GLP-1R were resuspended in activation solution at a density of 6 × 10⁵ / 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 Eu-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 GLP-1. The percentage of maximum response was compared with the concentration of the RNA transfected cell supernatant (2 μg of circular RNA transfected into cells was set as the initial concentration of 1M, followed by 5-fold dilutions for a total of 8 gradients).

[0320] The data were transformed using the cAMP standard curve and analyzed using a three-parameter logistic equation. The results are shown in Table 12.

[0321] Table 12. Agonistaltic activity of exendin-4 expressed by circRNA on GLP-1 receptor

[0322] Example 5: In vivo efficacy evaluation of circular RNA formulation in diet-induced obese C57BL / 6 mice.

[0323] To investigate the effects of the circular RNA formulations from Example 3 on parameters such as weight loss, metabolism, body composition, and hepatic steatosis, they were administered to C57BL / 6 diet-induced obese (DIO) mice. These mice were older than 20 weeks after being placed on a high-fat diet for more than 12 weeks. Specifically, 24- to 25-week-old male DIO C57BL / 6 mice dependent on a high-calorie diet were used in the following studies. Mice were individually housed in a temperature-controlled facility (20°C to 26°C) with a 12-hour light / 12-hour dark diurnal cycle provided by a time-controlled lighting system (light hours from 7:00 AM to 7:00 PM). They had free access to food (D12492 (Research diets)) and water. DIO mice were selected based on baseline weight data and randomly assigned to groups of 5-6 mice each, weighing ≥40g. On day -1, weight and body composition were measured, and all mice were regrouped into 7 groups based on weight. On day 0, fasting blood glucose was measured (overnight fasting). On day 1, treatment began, and body weight and 24-hour food intake were measured three times a week. On day 28, body composition was measured. On day 29 (4 weeks), animals were fasted overnight. a) Fasting blood glucose was measured, and no medication was administered on the last day. b) Animals were euthanized by CO2. c) Blood was collected into heparin tubes. Blood was centrifuged at 4500 rpm for 10 minutes at 4°C, and plasma was collected. d) Liver was collected and photographed, and liver weight was measured. e) Epididymal fat, subcutaneous inguinal fat, mesenteric fat, perirenal fat, and brown fat were collected and weighed. Plasma biochemical indicators (endpoints): total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT).

[0324] Frozen liver tissue was thawed at room temperature for 30 min (until the liver was soft), then 1.3 mL of lysis buffer (anhydrous ethanol: 30% KOH solution = 2:1 volume ratio) was added, and the tissue was incubated overnight at 55°C. After complete lysis, the liver tissue was centrifuged at 4°C and 4000 rpm for 10 min. The supernatant was then collected and the concentrations of LDL-C and TC were determined using a BioMajesty portable biochemical analyzer.

[0325] After blood collection from the animals, they were dissected to obtain subcutaneous fat from the abdomen, epididymal fat, perirenal fat, and brown fat from the scapula. The fat was weighed separately, and the organ coefficients were calculated.

[0326] Data are presented as mean ± standard error (Mean ± SEM) for 5-6 animals per group, as shown in the table below. Experimental data were statistically analyzed using one-way ANOVA. If the ANOVA was statistically significant (P < 0.05) and the variances were homogeneous, the Tukey test was used for inter-group comparisons. If the variances were unequal, the Dunnet's T3 test was used. A P < 0.05 was considered statistically significant. This experiment statistically analyzed and reported the differences between the solvent control group and each drug administration group, the positive control and the test product equal-dose group, and different dose groups of the test product or positive control.

[0327] Table 13 Weight changes after 28 days following treatment with the analogue.

[0328] *: p < 0.05; **: p < 0.01; ***: p < 0.001 compared with the PBS negative control group.

[0329] #:p<0.05; ##:p<0.01; ###:p<0.001 compared with the LNP empty negative control group.

[0330] Compared to the PBS group (Group 1), the body weight percentage (BGS) in the 0.4 mpk Exenatide (three times weekly) group significantly decreased on days 3 and 8–27; the BGS in the 0.4 mpk CR026-01 (once weekly) group significantly decreased on days 3–27; and the BGS in the 0.4 mpk CR026-02 (once weekly) group significantly decreased on days 6–27. Compared to the LNP control group (Group 3), the BGS in the 0.4 mpk CR026-01 (once weekly) group significantly decreased on days 6–27; and the BGS in the 0.4 mpk CR026-02 (once weekly) group significantly decreased on days 6–27. CR026-01 uses circular RNA to express Exendin-4, demonstrating that circular RNA technology may provide longer duration of drug action, higher stability, and greater efficacy compared to conventional Exenatide peptide drugs. This form of expression can prolong the drug's half-life and reduce the frequency of administration, for example, from multiple injections per week (e.g., three times a week) of peptide drugs to once a week or longer. The weight loss effect of CR026-01 also demonstrates the potential of the circular RNA form in terms of pharmacological efficacy in experiments.

[0331] Table 14-1 Changes in body fat percentage after 28 days following treatment with the analogue.

[0332] Table 14-2 Changes in body fat percentage after 28 days following treatment with the analogue.

[0333] On day 28, compared with the PBS group, the 0.4 mpk Exenatide (three times a week) group and the 0.4 mpk CR026-01 (once a week) group showed significant reductions in fat mass and fat percentage. Compared with the LNP control group, the 0.4 mpk CR026-01 (once a week) group showed a significant reduction in fat mass. Both the CR026-01 (once a week) group and the Exenatide (three times a week) group showed significant reductions in body fat, with marked differences compared to the PBS group, indicating that the circular RNA-expressed Exendin-4 has a similar effect on reducing body fat as the peptide form of Exenatide.

[0334] On day 28, there was no statistically significant difference in lean body mass compared to the PBS group or the LNP control group. On day 28, compared to the PBS group (Group 1), the lean body mass percentage in the 0.4 mpk CR026-01 (once weekly) group was significantly increased due to weight loss. The significantly higher lean body mass percentage in the CR026-01 (once weekly) group indicates an increased lean body mass percentage, which may be related to weight loss and fat reduction. However, there was no significant difference in lean body mass percentage between the Exenatide (three times weekly) group and the PBS group, suggesting that the three-times-weekly peptide formulation is relatively less effective in maintaining lean body mass.

[0335] CR026-01 showed better results in reducing body fat and increasing lean body mass percentage, especially in lean body mass, demonstrating a more significant advantage over the peptide form administered three times weekly. This suggests that circular RNA technology may have better potential for improving body composition, while requiring less frequent administration.

[0336] Table 15-1 Changes in organ weight 28 days after treatment with the analogue of the example.

[0337] Table 15-2 Changes in organ weight 28 days after treatment with the analogue of the example.

[0338] On day 29, the animals were euthanized, and the livers were weighed and collected. At the final point, no significant differences were found in liver weight or liver index. On day 29, adipose tissue was weighed and collected. The inguinal adipose tissue weight was significantly reduced in the 0.4 mpk CR026-01 (once weekly) group compared to the PBS group. White fat weight was calculated as the sum of epididymal fat, mesenteric fat, inguinal fat, and perirenal fat. The white fat weight was significantly reduced in the 0.4 mpk Exenatide (three times weekly) group compared to the PBS group. There were no significant differences in liver and epididymal fat weight between the groups.

[0339] Overall, the effect of exendin-4 (CR026-01, expressed via circular RNA, once weekly) on reducing adipose tissue (including epididymal, mesenteric, inguinal, and perirenal fat) was comparable to that of exenatide (three times weekly), and may have been more significant in reducing subcutaneous adipose tissue. However, there was no significant difference in their effects on liver weight.

[0340] Table 16-1 Effects of treatment with the analogue on blood glucose, cholesterol, and triglycerides after 28 days.

[0341] Table 16-2 Effects of treatment with the analogue on blood glucose, cholesterol, and triglycerides after 28 days.

[0342] Serum was collected on day 29 after an overnight fast. Compared with the PBS group, the 0.4 mpk CR026-01 (once weekly) group showed a significant decrease in serum total cholesterol (TC). Compared with the LNP control group, the 0.4 mpk CR026-01 (once weekly) group also showed a significant decrease in serum TC. Compared with the LNP group, the 0.4 mpk CR026-01 (once weekly) group showed a significant decrease in serum total triglycerides (TG). No decreasing trend was observed in serum low-density lipoprotein cholesterol (LDL-c), alanine aminotransferase (ALT), aspartate aminotransferase (AST), fasting liver TC, and TG at the endpoints, but these differences were not statistically significant. There were no differences in fasting blood glucose among the groups, possibly because the fasting blood glucose in the negative control group was already low (8.57 ± 0.40 mmol / L), close to the normal range. For individuals with low blood glucose, the hypoglycemic effect of the drug may not be significant. In such cases, more severe diabetes models (such as STZ-induced type 1 diabetes models or db / db mouse type 2 diabetes models) are often more suitable for evaluating drug efficacy.

[0343] Overall, the 0.4mpk CR026-01 (once a week) group was more effective than the 0.4mpk Exenatide (three times a week) group in lowering total plasma cholesterol.

[0344] Example 6: Preparation of LNP formulations with different prescriptions

[0345] This study aimed to encapsulate circular RNA (CR053-FPE) using two different LNP formulations, comparing key physicochemical indicators such as particle size, PDI, and encapsulation efficiency of the LNPs from different formulations. The encapsulated samples obtained will be used in subsequent animal experiments to comprehensively evaluate the performance of each formulation and screen for the most promising formulation. In this experiment, except for the LNP formulation, the batches of circular RNA, encapsulation equipment, and processes were kept consistent to ensure the reliability and comparability of the experimental results.

[0346] 6.1 Experimental Materials

[0347] The ionizable lipid molecule SM-102, an auxiliary phospholipid (DSPC), was purchased from Corden Pharma; cholesterol was purchased from Sigma-Aldrich; and mPEG2000-DMG (i.e., DMG-PEG 2000 or PEG-DMG2K) was purchased from Avanti Polar Lipids, Inc. The ionizable lipid molecule HL1001 was purchased from Guangzhou Hengnuokang Pharmaceutical Technology Co., Ltd.

[0348] 6.2 Preparation of circular RNA (CR053-FPE)

[0349] Plasmid preparation: The target sequence is the same as in section 1.2 of Example 2. The plasmid synthesized by GenScript was transformed into the stbl3 strain. 20 μL of competent cells were taken, 0.5 μL of plasmid was added, and the mixture was incubated on ice for 30 minutes, then in a 42°C water bath for 90 seconds. 1 mL of culture medium was added, and the mixture was then placed in a 37°C, 250 rpm shaker for 60 minutes. 100 μL of the culture was then dropped onto an agar plate, spread evenly, air-dried, and incubated overnight upside down. The plate was removed from the incubator, and single colonies were picked for bacterial amplification. The strain was then cryopreserved. 15 mL of LB medium containing 0.1% kanamycin was dispensed into a 125 mL sterile shake flask and incubated in a 37°C shaker for 15 minutes for preheating.

[0350] After thawing, the entire CR053-FPE frozen strain was added to 15 mL of culture medium and cultured at 37°C in a shaker for 6 hours to prepare the seed culture. 1 L of LB medium containing 0.1% kanamycin was dispensed into each of four 3 L sterile shake flasks and preheated at 37°C for 15 min in a shaker. In a clean bench, the seed culture was inoculated into the preheated LB medium at a ratio of 1:100 and cultured overnight at 37°C in a shaker. The overnight fermentation broth was centrifuged at 8000 x g for 10 min, and the supernatant was discarded.

[0351] Extract plasmids using the Nucleobond Endotoxin-Free Plasmid Extraction Kit. Take 20g of wet bacterial cells and divide them into two portions. Add 120mL of buffer S1 to each aliquot and thoroughly resuspend the bacterial cell pellet using a pipette or vortex mixer. Add 120mL of buffer S2 to each aliquot, invert 4-6 times to mix thoroughly, and incubate at room temperature (15-25℃) for 5 minutes. Add 120mL of buffer S3 to each aliquot, immediately invert 4-6 times to mix thoroughly. Pour the lysis buffer into the filter cartridge and incubate at room temperature for 5 minutes. Add 100mL of buffer N2 to each PC 10000EF column and allow the column to empty under gravity. Add the lysate obtained from the previous filtration onto a PC 10000EF column, allowing it to flow into the adsorption column by gravity. Wash each PC 10000EF column with 4 x 150 mL buffer N3 and 3 x 130 mL buffer N4. Elute the plasmid with 100 mL buffer N5. Precipitate the DNA by adding 70 mL (0.7 CV) of room temperature isopropanol to each eluted DNA. Mix and immediately centrifuge at 4°C at >15000 xg for 30 minutes. Carefully discard the supernatant. Wash each DNA precipitate with 5 mL of endotoxin-free 70% ethanol at room temperature (add 40 mL of 96–100% ethanol to the endotoxin-free water provided in the kit) and centrifuge at 15000 xg for 10 minutes. Carefully pour out the supernatant, air dry the precipitate, and collect all the plasmid precipitate in the tube into a single tube. Redissolve the precipitate in 10 mL of water for injection, and then purify and concentrate it to a final volume of 10 mL using a 100 KD hollow fiber column.

[0352] Linearization template preparation: The CR053-FPE plasmid was linearized by EcoR I restriction endonuclease to prepare an IVT transcription template. The standard enzyme digestion formulation is shown in Table 17.

[0353] Table 17 Standard Formulation of EcoR I Enzyme Digestion System (1ml)

[0354] Prepare one 12 mL reaction system by scaling up the standard formulation system in Table 17. Place the reaction system in a metal water bath and react at 37°C for 5 hours. After the reaction is complete, terminate the enzymatic digestion reaction at 65°C for 20 minutes. After digestion, remove the digestion system from the water bath and concentrate it by tangential flow filtration using a 100 kDa hollow fiber column. After 10 washes, collect the liquid. Add 1 / 20 volume of 0.5 M EDTA solution, 1 / 10 volume of 3 M sodium acetate solution, and 2 volumes of anhydrous ethanol to the digestion system. Mix well and place the digestion system in a -20°C freezer for 15 minutes. Then centrifuge at the highest speed of 16500 rpm (18360 × g) at 4°C for 15 minutes. Remove the supernatant, rinse the precipitate with 70% ethanol solution, and centrifuge again at 16500 rpm (18360 × g) at 4°C for 5 minutes. After completely aspirating the supernatant, add 10 mL of water for injection to dissolve the precipitate. Add 0.3M sodium acetate and 2 volumes of anhydrous ethanol to the purified product again, and repeat the ethanol precipitation purification step once more. Let the mixture stand at room temperature for at least 10 minutes to ensure complete evaporation of the ethanol in the precipitate. Add 9 mL of water for injection to dissolve the precipitate and obtain the linearized template. Samples were sent for concentration and HPLC purity testing (the purity of the linearized template was determined using AEX-HPLC, which distinguishes linearized plasmids from supercoiled plasmids by the different numbers of phosphate groups exhibited by different plasmid structures) to ensure that the prepared linearized template meets the requirements for subsequent transcription.

[0355] In vitro transcription: Prepare 50 mL IVT systems according to the standard in vitro transcription system in Table 18 below. Place the transcription systems in a constant temperature shaker at 47°C for 1 hour. After the reaction is complete, add 2.5 mL of DNase I and 5.75 mL of 10×DNase I buffer to each reaction system and incubate at 37°C for 15 minutes. Then, add 1.02 mL of 500 mM EDTA to terminate the reaction.

[0356] Table 18 Standard System for In Vitro Transcription

[0357] Purification: The crude IVT reaction solution was diluted to a total volume of 250 mL. The crude circRNA reaction solution obtained from in vitro transcription was then replaced using tangential flow filtration (TFF) to remove NTPs and most proteases from the IVT process. 0.11 mg / mL of urea solution was used. 2A 100KD membrane was used, with water for injection as the replacement solution. During the replacement process, water for injection was continuously injected into the storage tank at a constant volume, while maintaining a balance between the filtrate flow rate and the influent flow rate to keep the liquid level in the tank stable. The replacement process was stopped when the filtered volume reached four times the tank volume, i.e., 1L. Afterwards, the crude RNA product obtained after the replacement was purified using a Seppic SCG-P 100 protein purification system via reverse-phase chromatography (the crude circRNA product was purified using reverse-phase chromatography, distinguishing between circRNA and nick RNA by the different binding degrees of ion-pairing reagents). The target circRNA peak was collected, and the final circRNA product was obtained after the replacement process, which was then used for subsequent encapsulation.

[0358] 6.3 Preparation of Circular RNA-LNP Encapsulated Samples

[0359] circRNA dilution: Dilute CR053-FPE Mix with an aqueous phase of Water 240402 to an N / P ratio of 6.

[0360] For the LNP-01 formulation, the four lipid components were accurately weighed according to the molar ratio of SM-102:cholesterol:DSPC:mPEG-DMG-2K = 50:10:38.5:1.5, and dissolved in anhydrous ethanol to prepare a lipid solution with a total lipid concentration of 16mM. The batch number of this solution is HUO250324.

[0361] For the LNP-03 formulation, the four lipid components were accurately weighed according to the molar ratio of HL1001:cholesterol:DSPC:mPEG-DMG-2K = 50:10:38.5:1.5, and dissolved in anhydrous ethanol to prepare a lipid solution with a total lipid concentration of 16mM. The batch number of this solution is HL1001-250326.

[0362] Following the instructions for using the microfluidic preparation system, in the initial stage of preparation, since the system has not yet reached a stable state, a suitable amount of waste liquid needs to be discarded to ensure that the subsequent encapsulation process is carried out under stable conditions. During the experiment, the total flow rate was set to 60 mL / min, the flow rate ratio of the aqueous phase to the lipid phase was 3:1, and the online dilution factor was set to 5 times. For two different lipid phase formulations (LNP-01 and LNP-03), microfluidic mixing was performed separately, resulting in encapsulation intermediates with batch numbers CR059 LSM250325MF01N and CR059 LHN250326MF02N, respectively.

[0363] The two encapsulated intermediates obtained by the above microfluidic preparation were subjected to tangential flow ultrafiltration (TFF) for liquid exchange and concentration. First, the encapsulated intermediates were subjected to TFF ultrafiltration using CCS250321 solution, with a washing volume of 2 times; then, the solution was switched to TBS250321 solution, and the washing volume of 4 times was continued.

[0364] After the solution exchange, the sample solution was concentrated to approximately 40 mL using a TFF system. One-quarter volume of 40% Sucrose 250 325F solution was slowly added to the concentrated sample solution, and after thorough mixing, it was filtered through a 0.22 μm sterile filter membrane for sterilization. The solution was then aliquoted into sterile cryovials at 1 mL / vial. The batch numbers corresponding to the two formulations (LNP-01 and LNP-03) are CR059-DP250325 and CR059-DP250326B, respectively.

[0365] Table 19 Quality test results of two types of circular RNA encapsulated samples

[0366] The quality test results of the two circular RNA encapsulated samples (CR059-DP250325 and CR059-DP250326B) are shown in Table 19. Both samples meet the sample quality release standards and can be used for subsequent cell experiments and other studies.

[0367] 6.4 Protein Expression Detection

[0368] Cell preparation: HEK293T cells with 90% confluence were collected, the cell culture medium was discarded, and the cells were washed with PBS. The PBS was discarded, and the cells were digested with 0.25% trypsin. After the cells became rounded, an appropriate amount of antibiotic-free cell culture medium (DMEM containing 10% FBS) was added, and the cells were pipetted several times. The cells were then transferred to a 50mL centrifuge tube, centrifuged at 800rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended with an appropriate amount of antibiotic-free cell culture medium. Cell counting was performed, and the cell concentration was adjusted to 1×106 cells / mL. 1mL of the cell culture medium was seeded into 12-well plates, and the cell culture plates were then incubated at 37℃ in a 5% CO2 incubator for about 24 hours.

[0369] Transfection: For CR059-DP250325 and CR059-DP250326B cells, 200 ng of LNP sample was added dropwise to each well. Cells seeded the previous day were removed from the incubator, and the above transfection mixture was added dropwise to the seeded cells. After gentle mixing, the cells were incubated at 37°C in a 5% CO2 incubator for approximately 24 h, 72 h, and 168 h. Negative control: HEK293T cells not transfected with LNP sample were used as a blank control group.

[0370] Supernatant protein collection: At 24h, 72h and 168h after transfection, the cell culture plate was removed from the CO2 incubator, and the supernatant was collected into a 1.5mL centrifuge tube. The tube was centrifuged at 1000g at 4℃ for 10 minutes to remove cell debris. The supernatant was then collected into a new 1.5mL centrifuge tube and stored at -80℃.

[0371] ELISA assay: The experiment was performed according to the manufacturer's instructions (Exendin-4 ELISA kit, Abcam, UK).

[0372] Table 20 Exendin-4 ELISA Detection Results

[0373] Based on the experimental results, the expression levels of the three samples screened for the prescription showed that, at the average expression level, CR059-DP250325 (SM-102 lipid) ≈ CR059-DP250326B (HL1001 lipid). Hereafter, CR059-DP250326B (HL1001 lipid) will be referred to simply as CR059.

[0374] Example 7: Efficacy evaluation of circular RNA formulation in diabetic db / db mice

[0375] The pharmacodynamics of the test substance CR059 (RNA CR053-FPE, LNP LNP-03, i.e., cation HL1001) was evaluated in db / db mice via subcutaneous injection, with the marketed similar product Semaglutide as a positive control.

[0376] Group 1 received a subcutaneous injection of the solvent; Group 2 received a subcutaneous injection of CR057-BK (empty LNP); Groups 3–6 received subcutaneous injections of CR059 at concentrations of 3 μg / kg, 10 μg / kg, 30 μg / kg, 90 μg / kg, 180 μg / kg, and 360 μg / kg, respectively; and Group 7 received a subcutaneous injection of semaglutide at 120 μg / kg. The administration volume was 5 mL / kg, calculated based on body weight on the day of administration (QW*2). The first administration was recorded as Day 1.

[0377] Random blood glucose testing: twice during the adaptation period (Day 7 and Day 3). Four times during the treatment period (0h before the first dose, 8h, 24h, and 48h after the first dose). Fasting blood glucose testing: once during the adaptation period, 24h after the second dose.

[0378] Table 21: Random blood glucose-lowering effect of CR059 (CR053-FPE) in db / db mice and its comparison with semaglutide.

[0379] *: p < 0.05; **: p < 0.01; ***: p < 0.001 compared with the vehicle negative control group

[0380] In db / db mice, CR059 exhibited a significant dose-dependent hypoglycemic effect. Following a single dose, CR059 reduced random blood glucose levels across a range of 3–360 μg / kg, with the hypoglycemic effect increasing with increasing dose. Specifically, a dose of 30 μg / kg reduced blood glucose to 30.4 ± 0.6 mmol / L 24 hours after administration, a decrease of approximately 9% from baseline. At doses of 90 μg / kg and above, the hypoglycemic effect was more pronounced, with 24-hour blood glucose levels decreasing to 21.8 ± 3.0 mmol / L (90 μg / kg), 20.1 ± 2.4 mmol / L (180 μg / kg), and 18.6 ± 2.6 mmol / L (360 μg / kg), respectively. In contrast, semaglutide at a dose of 120 μg / kg only reduced blood glucose to 25.0 ± 1.4 mmol / L 24 hours after administration.

[0381] The above results indicate that CR059 exhibits comparable hypoglycemic effects at relatively low doses (30 μg / kg), while at higher doses (90–360 μg / kg), its hypoglycemic effect is superior to that of Semaglutide (120 μg / kg). These results suggest that CR059 possesses stronger hypoglycemic efficacy and good dose-dependency, demonstrating pharmacodynamic characteristics superior to Semaglutide.

[0382] Table 22. Effects of CR059 on improving fasting blood glucose in db / db mice and its comparison with semaglutide.

[0383] *: p < 0.05; **: p < 0.01; ***: p < 0.001 compared with the vehicle negative control group

[0384] In db / db mice, CR059 significantly reduced fasting blood glucose in a clear dose-dependent manner. A single dose of 3–30 μg / kg slightly improved fasting blood glucose, while the hypoglycemic effect was particularly pronounced at doses of 90 μg / kg and above. Specifically, a 90 μg / kg dose reduced fasting blood glucose to 18.4 ± 3.1 mmol / L 24 hours after administration, a decrease of approximately 37% from baseline; a 180 μg / kg dose reduced it to 20.0 ± 2.9 mmol / L; and at 360 μg / kg, blood glucose was further reduced to 14.1 ± 1.6 mmol / L.

[0385] In contrast, semaglutide at a dose of 120 μg / kg only decreased to 27.4 ± 1.9 mmol / L after 24 hours. These results indicate that CR059 at medium to high doses (≥90 μg / kg) is significantly more effective than semaglutide (120 μg / kg), demonstrating stronger efficacy and good dose-dependency.

[0386] Example 8: Efficacy evaluation of circular RNA formulation in spontaneously diabetic rhesus monkeys

[0387] To investigate the efficacy and safety of the circular RNA formulation CR059 (RNA CR053-FPE, LNP LNP-03, i.e., cation HL1001) administered twice subcutaneously in spontaneously diabetic type 2 rhesus monkeys, and to provide dosage design basis and safety information for subsequent animal and clinical trials. Inclusion criteria for spontaneously diabetic type 2 rhesus monkeys: male / female, age 12-20 years (equivalent to 36-60 years of adulthood); FPG: 5.5-8.0 mmol / L; HbA1c ≥ 4.5%;

[0388] Table 23 Comparison of blood glucose levels in humans and rhesus monkeys under different conditions.

[0389] Note: The blood glucose range data for rhesus monkeys were obtained from Sichuan Prame Biotechnology Co., Ltd., and were measured under anesthesia.

[0390] Four spontaneously diabetic rhesus monkeys were enrolled in the experiment and divided into two groups. Fasting plasma glucose (FPG) and glycated hemoglobin (HbA1c) were measured once during the animal adaptation / baseline period (within 2 weeks before grouping).

[0391] Table 24 Group Design Information

[0392] Two tests were conducted during the adaptation period and baseline period within the first two weeks of grouping. Blood samples were collected once each before the first administration (0h) and at 24h, 72h, 120h, and 168h after administration, and then weekly thereafter. The second injection was administered on day 32. Fasting blood glucose was measured on days 1 and 32 before administration. Administration method: Each animal received a subcutaneous injection in the upper limb, with the injection site shaved before a single-point injection.

[0393] Table 25-1 Summary of fasting blood glucose parameters of CR059 in spontaneously diabetic rhesus monkeys

[0394] Table 25-2 Summary of fasting blood glucose parameters of CR059 in spontaneously diabetic rhesus monkeys

[0395] Table 26-1 Summary of efficacy and safety indicators of CR059 in spontaneously diabetic rhesus monkeys

[0396] Table 26-1 Summary of efficacy and safety indicators of CR059 in spontaneously diabetic rhesus monkeys

[0397] CR059 demonstrated significant hypoglycemic activity and good safety in spontaneously diabetic rhesus monkeys. Results showed that CR059 at doses of 20–60 μg / kg effectively reduced fasting plasma glucose (FPG) and HbA1c levels in a dose-dependent manner. In the high-dose group (60 μg / kg), body weight decreased slightly during the experiment, but blood glucose remained at a low level, with FPG decreasing from 6.20–6.31 mmol / L to 6.12–6.15 mmol / L and HbA1c decreasing from 4.83–4.95% to 4.34–4.40%. The low-dose group (20 μg / kg) also showed improved blood glucose levels, with FPG decreasing from 5.71–6.10 mmol / L to 5.02–5.69 mmol / L and HbA1c decreasing from 4.77–4.97% to 4.10–4.33%. Meanwhile, liver and kidney function indicators (ALT, AST), blood lipids, and other safety indicators remained within the normal range, suggesting that CR059 has good tolerability and safety. In summary, CR059 demonstrates significant clinical application potential in spontaneously diabetic rhesus monkeys by combining rapid glucose reduction, controllable dose-dependent effects, stable blood glucose maintenance, and good safety.

Claims

1. A recombinant protein comprising a GLP-1 receptor agonist, wherein the GLP-1 receptor agonist is selected from at least one of SEQ ID NO: 17, 19, 23-26, and a sequence having more than 90% identity to achieve the same function, and wherein one or more amino acid residues are substituted, added, inserted or deleted in the above sequence. Optionally, the recombinant protein comprises a signal peptide; Optionally, the recombinant protein comprises a self-cleaving peptide selected from any one of SEQ ID NO:20-22.

2. The recombinant protein according to claim 1 may be as shown in SEQ ID NO:3 or 16, or a sequence that achieves the same function with more than 90% identity, or a sequence in which one or more amino acid residues are substituted, added, inserted or deleted.

3. A nucleic acid molecule capable of expressing the recombinant protein of claim 1; preferably, the nucleic acid molecule may be a DNA molecule or an RNA molecule; optionally, the DNA molecule may be selected from SEQ ID NO:1, 4, 6, 8, 10, 12, 14, the RNA molecule may be selected from SEQ ID NO:2, 5, 7, 9, 11, 13, 15, and sequences that achieve the same function with more than 90% identity, and sequences in which one or more amino acid residues are substituted, added, inserted or deleted, preferably 1-3 residues.

4. An expression vector capable of producing the recombinant protein of claims 1-2, and capable of containing the nucleic acid molecule of claim 3.

5. The expression vector according to claim 4, comprising linear precursor RNA, wherein the linear precursor RNA molecule comprises homologous arms, exons, introns, spacers, internal ribosome entry sites, and a coding region; further, the structure may be as follows: 5' outer homologous arm - IG5 - 5' intron - 3' intron - E2 - 5' spacer - Internal ribosome entry site (IRES) - coding region - 3' spacer - E1 - IG3 - 3' outer homologous arm or 5' outer homologous arm - 3' intron - 5' spacer region - internal ribosome entry site (IRES) - coding region - 3' spacer region - 5' intron - 3' outer homologous arm.

6. The expression vector according to claim 5, comprising a circulatory element with RNA sequence SEQ ID NO: 27-37, or a circulatory element with RNA sequence SEQ ID NO: 49-55; further, the corresponding circulatory element comprising DNA sequence SEQ ID NO: 38-48, or a circulatory element comprising DNA sequence SEQ ID NO: 56-62, any one of which, and a sequence having more than 90% identity to achieve the same function, and one or more amino acid residues substituted, added, inserted, or deleted in the above sequences.

7. The expression vector according to claim 6 may be a linear precursor RNA molecule as shown in SEQ ID NO: 63-69, and a DNA molecule as shown in SEQ ID NO: 70-76 expressing the linear precursor RNA molecule, and any one of the sequences having an identity of more than 90% that can achieve the same function, and one or more nucleotide residues may be substituted, added, inserted or deleted in the above sequences.

8. The expression vector according to claim 4, wherein the expression vector is preferably a circular RNA molecule, the sequence of which may be as shown in SEQ ID NO: 77-83, any one of which and any sequence having more than 90% identity to achieve the same function, and one or more nucleotide residues may be substituted, added, inserted or deleted in the above sequence.

9. A host cell capable of containing the expression vector according to any one of claims 4-9.

10. A composition comprising the circular RNA molecule of claim 8; preferably, the composition comprises lipids encapsulating the circular RNA molecule; the lipids comprising LNP, liposomes, or exosome delivery systems.

11. The composition according to claim 10, wherein the LNP comprises a cationic lipid or ionizable lipid selected from SM-102, ALC-0315, Dlin-MC3-DMA, DODMA, C12-200, DlinDMA, HL1001, an accessory lipid selected from DSPC, DOPE, DOPC, DOPG, or DOPS, cholesterol or a cholesterol derivative, and a PEG lipid selected from mPEG2000-DMG, DSG-PEG2000, or ALC-0519.

12. The composition according to claim 12, wherein the LNP composition preferably has a molar ratio of SM102 and / or HL1001:cholesterol:DSPC:mPEG-DMG-2K of (25-65):(25-70):(5-25):(0.5-5), preferably (23-61):(27-66):(8-22):(0.7-4), or further, (27-58):(30-60):(8-19):(0.7-3.5), more preferably (30-55):(30-50):(8-16):(0.7-2.0).

13. The composition according to claim 11, wherein the concentration of the lipid solution can be between 5 mg / mL and 15 mg / mL, specifically 5-10 mg / mL, more preferably 5 mg / mL, 7 mg / mL, 10 mg / mL, 12 mg / mL, or 15 mg / mL, and even more preferably selected from 7 mg / mL, 10 mg / mL, or 12 mg / mL.

14. Use of the recombinant protein of claims 1-2, the nucleic acid molecule of claim 3, the expression vector of claims 4-8, the host cell of claim 9, and the composition of claims 10-13 in the treatment and prevention of metabolic diseases, and in the preparation of medicaments for the treatment of metabolic diseases.

15. The use of the recombinant protein of claims 1-2, the nucleic acid molecule of claim 3, the expression vector of claims 4-8, the host cell of claim 9, and the composition of claims 10-13 in assisting weight loss, and in the preparation of food and health products with weight loss function.

16. Use of the recombinant protein of claims 1-2, the nucleic acid molecule of claim 3, the expression vector of claims 4-8, the host cell of claim 9, and the composition of claims 10-13 in regulating insulin, blood glucose, cholesterol, and triglyceride levels in patients.

17. The use according to claim 14, wherein the metabolic disease includes diabetes, obesity, dyslipidemia, elevated glucose levels, elevated insulin levels, and diabetic nephropathy, etc.

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