A method for preparing smegglutinin using circRNA and related compositions

Abstract

This invention relates to the field of biopharmaceutical technology, and discloses a method for preparing smegglutinin using circRNA and related compositions. The circRNA molecule contains an open reading frame and an internal ribosome entry site sequence. A recombinant plasmid containing a T7 promoter, introns, and coding sequences is constructed and linearized. A linear RNA intermediate is obtained through in vitro transcription; subsequently, it is placed in a circularization buffer for self-splicing and circularization; and purified to obtain the circRNA molecule stock solution. The related pharmaceutical composition contains circRNA molecules, a four-component lipid mixture, and excipients containing carbohydrates and nonionic surfactants. This invention constructs circRNA molecules with closed-loop structures and utilizes the internal ribosome entry site sequence to initiate translation. The lack of ends in the closed-loop structure avoids exonuclease degradation, thus prolonging the protein expression cycle, improving the purity of the stock solution, and reducing the risk of immune responses caused by linear RNA impurities.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a method for preparing smegglutinin using circRNA and related compositions. Background Technology

[0002] Smegglutide is a glucagon-like peptide-1 receptor agonist with a high degree of homology to human endogenous glucagon-like peptide-1. It mainly controls blood sugar and regulates weight through mechanisms such as promoting insulin secretion from pancreatic β cells, inhibiting glucagon release from pancreatic α cells, delaying gastric emptying, and suppressing appetite. It is currently used to treat type 2 diabetes and obesity.

[0003] Current technologies for preparing smegglutinin mainly rely on chemical synthesis or recombinant protein expression, but these methods have certain limitations. Chemical synthesis typically employs solid-phase peptide synthesis technology, requiring multiple amino acid coupling, deprotection, and cleavage reactions, making the process cumbersome and prone to producing impurities such as deleted peptides or epimeric peptides, resulting in high production costs. While recombinant protein expression utilizes biological synthesis, E. coli expression systems are prone to inclusion body formation, limiting refolding rates; and although mammalian cell expression systems possess modification capabilities, they have long culture periods, and the products are easily degraded by intracellular proteases. Furthermore, directly administered peptide drugs have short half-lives in vivo, often requiring frequent dosing to maintain effective blood drug concentrations, impacting patient compliance.

[0004] Circular RNA (cRNA), a type of nucleic acid molecule with a closed circular structure, exhibits resistance to exonuclease degradation due to the lack of a 5' cap and a 3' polyadenylate tail, and possesses the potential for sustained protein expression in vivo. However, during the in vitro preparation of cRNA, the cyclization reaction often results in the residue of uncirculated linear RNA precursors and splicing byproducts. If these linear impurities are not effectively removed, they can easily induce innate immune responses in the body, affecting the safety and purity of the drug. Furthermore, the delivery of cRNA into the body requires carriers such as lipid nanoparticles, but existing preparation processes struggle to precisely control particle size uniformity when mixing organic and aqueous phases. Moreover, the prepared lipid nanoparticles are prone to aggregation or fusion during storage, leading to decreased physicochemical stability of the drug composition. Therefore, how to prepare high-purity cRNA molecules encoding semaglutide and construct a delivery system with good uniformity and storage stability is a problem that needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing semaglutide using circRNA and related compositions, which solves the problems of limited in vivo expression cycle of existing semaglutide drugs, the risk of immune response caused by linear impurities during circRNA preparation, and poor particle size uniformity and insufficient storage stability of lipid nanoparticle delivery systems.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides a circRNA molecule for preparing smegglutinin, using the following technical solution: A circRNA molecule for preparing semaglutide, the circRNA molecule comprising an open reading frame encoding a semaglutide precursor protein and an internal ribosome entry site sequence; wherein the internal ribosome entry site sequence is selected from encephalomyocarditis virus IRES, the nucleotide sequence of which is shown in SEQ ID NO:5; the amino acid sequence of the semaglutide precursor protein is shown in SEQ ID NO:4, and the semaglutide precursor protein comprises, from the N-terminus to the C-terminus,: a signal peptide, the amino acid sequence of which is shown in SEQ ID NO:2; a linker peptide, the amino acid sequence of which is shown in SEQ ID NO:3; and a mature semaglutide peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] By employing the above technical solution, the closed-loop circRNA molecule, lacking a 5' cap and a 3' polyadenylated tail, can circumvent the recognition and degradation by intracellular exonucleases, thus enhancing the intracellular stability of the RNA molecule and prolonging the expression cycle of the smegraglutide protein. Simultaneously, utilizing the internal ribosome entry site sequence derived from encephalomyocarditis virus, ribosomes can directly bind to the RNA to initiate translation, achieving cap-independent translation of the relevant protein. Furthermore, the smegraglutide precursor protein structure includes the signal peptide and the linker peptide. The signal peptide guides the translated protein into the secretory pathway, while the linker peptide facilitates the folding of the smegraglutide precursor protein and its subsequent in vivo processing and maturation, ensuring the biological activity of the expressed product.

[0008] Preferably, the circRNA molecule is prepared by the following steps: constructing a recombinant plasmid containing a T7 promoter, the 5' end of the Anabaena intron, the internal ribosome entry site sequence, the smegglutinin precursor protein coding sequence, and the 3' end of the Anabaena intron; linearizing the plasmid with restriction endonuclease and recovering the plasmid to obtain a linear DNA template; performing an in vitro transcription reaction using the linear DNA template to obtain a linear RNA intermediate; placing the linear RNA intermediate in a cyclization buffer containing GTP and MgCl2 for a self-splicing cyclization reaction to obtain a cyclization product; purifying the cyclization product to remove the uncyclized linear RNA precursor and splicing byproducts, and obtaining the circRNA molecule stock solution after purification.

[0009] By adopting the above technical solution, the in vitro automatic cyclization is achieved by utilizing the self-splicing mechanism of type I introns under the action of GTP and magnesium ions, without the need for additional ligases, thus reducing reaction costs.

[0010] Preferably, the restriction endonuclease linearization treatment is performed using XbaI restriction endonuclease at 36°C to 40°C for 2 to 4 hours; the in vitro transcription reaction is controlled at 36°C to 40°C for 2 to 4 hours.

[0011] By adopting the above technical solution and controlling the temperature and time of linearization and transcription, the DNA template cleavage effect and RNA transcription yield are guaranteed, the generation of non-specific products is reduced, and a substrate basis is provided for subsequent cyclization reactions.

[0012] Preferably, the concentration of GTP in the cyclization buffer is 1.5 mmol / L to 2.5 mmol / L, and the concentration of MgCl2 is 10 mmol / L to 30 mmol / L; the self-splicing cyclization reaction is controlled at a temperature of 50°C to 55°C and a reaction time of 15 minutes to 25 minutes.

[0013] By employing the above technical solution, the concentration ratio of GTP and MgCl2 provides a reaction environment for the self-splicing reaction of ribozymes. A reaction temperature of 50°C to 55°C can promote the formation of specific secondary structures in RNA molecules, thereby increasing cyclization efficiency by exposing active sites.

[0014] This invention also provides a semaglutide circRNA pharmaceutical composition, which adopts the following technical solution: A semaglutide circRNA pharmaceutical composition comprising: the circRNA molecule; a lipid mixture, by molar amounts, the lipid mixture comprising: 45 to 55 parts of positive lipids, 9.5 to 10.5 parts of phospholipids, 33.5 to 43.5 parts of cholesterol, and 1 to 2 parts of polyethylene glycol lipids; and excipients comprising carbohydrates and nonionic surfactants, wherein the final concentration of the carbohydrates is 5% g / v to 15% g / v, and the final concentration of the nonionic surfactants is 0.01% v / v to 0.1% v / v.

[0015] By employing the above technical solution, a specific four-component lipid nanoparticle delivery system can encapsulate negatively charged circRNA molecules. Specifically, the positively charged lipids protonate and bind to RNA in an acidic environment, reducing toxicity at physiological pH, and upon entering the cell, assist in RNA release through re-protonation. Cholesterol enhances the structural stability of the particles; phospholipids assist in the formation of a bilayer structure; and polyethylene glycol lipids form a hydration layer on the particle surface, inhibiting particle aggregation and reducing clearance by the reticuloendothelial system. The sugars in the excipients act as protectants or osmotic pressure regulators, preventing liposome fusion during storage; and the nonionic surfactants improve the colloidal stability of the system.

[0016] Preferably, the positive lipid is DLin-MC3-DMA; the phospholipid is DSPC; the polyethylene glycol lipid is PEG-DMG; the sugar is selected from sucrose or mannitol; and the nonionic surfactant is Tween 80.

[0017] By employing the above technical solution, DLin-MC3-DMA, as an ionizable positively charged lipid, possesses biodegradability and transfection efficiency, and together with the DSPC and PEG-DMG, constitutes a delivery carrier. The combination of sucrose and Tween 80 improves the physicochemical properties of the formulation, facilitating long-term storage and maintaining a uniform particle size distribution.

[0018] This invention also provides a method for preparing a semaglutide circRNA pharmaceutical composition, using the following technical solution: A method for preparing a semaglutide circRNA pharmaceutical composition includes the following steps: dissolving the lipid mixture in an organic solvent to obtain an organic phase solution; dissolving the circRNA molecules in an acidic buffer solution to obtain an aqueous phase solution; mixing the organic phase solution and the aqueous phase solution using a microfluidic chip system, collecting the eluent to obtain a crude lipid nanoparticle product; dialyzing the crude lipid nanoparticle product in a dialysis medium to remove the organic solvent and replace the buffer system to obtain a dialyzed lipid nanoparticle solution; adding the excipients to the dialyzed lipid nanoparticle solution to adjust the osmotic pressure and pH value, and filtering after sterilization to obtain the semaglutide circRNA pharmaceutical composition.

[0019] By employing the above technical solution, the mixing time and contact interface of the two-phase fluids can be controlled due to the use of microfluidic chip mixing technology. Within the microchannel, the organic phase solution and the aqueous phase solution are mixed, allowing lipid molecules to encapsulate the circRNA molecules during nucleation, thus preparing lipid nanoparticles with uniform particle size distribution and significant encapsulation efficiency. The dialysis step removes the organic solvent and replaces the buffer solution, ensuring the biocompatibility of the formulation.

[0020] Preferably, during the solution preparation process: the total lipid concentration of the organic phase solution is 10 mmol / L to 16 mmol / L; the mass concentration of the aqueous phase solution is 0.1 mg / mL to 0.3 mg / mL; and the acidic buffer solution is a citrate buffer solution with a pH of 3.8 to 4.2.

[0021] By employing the above technical solution, an environment with a pH of 3.8 to 4.2 is conducive to the full protonation of the positive lipids, which then bind to the circRNA molecules through electrostatic interactions, thereby improving the encapsulation efficiency. Controlling the ratio of lipid concentration to RNA concentration helps to form structurally stable nanoparticles with suitable charges.

[0022] Preferably, during the mixing process: the total flow rate of the microfluidic chip system during mixing is controlled to be 16 mL / min to 24 mL / min; the volume flow ratio of the aqueous phase solution to the organic phase solution is controlled to be 2:1 to 4:1.

[0023] By employing the above technical solution and controlling the volumetric flow rate ratio, the concentration of the organic solvent is diluted, thereby promoting the assembly of lipid molecules. The total flow rate controls the shear force and residence time of the fluid within the microchannel, maintaining the lipid nanoparticle structure while preventing particle aggregation and keeping the particle size within the range of 50 nm to 100 nm.

[0024] Preferably, during the dialysis and conditioning process: the volume of the dialysis medium is 100 to 150 times the volume of the crude lipid nanoparticle product, and the dialysis time is 10 to 14 hours; the buffer system is a phosphate buffer solution with pH 6.8 to pH 7.2; and the osmotic pressure of the regulated semaglutide circRNA drug composition solution is 280 mOsm / kg to 320 mOsm / kg.

[0025] By employing the above-mentioned technical solution, the residual amount of organic solvents in the dialysis process is reduced. Adjusting the pH to a neutral environment of 6.8 to 7.2 deprotonates the positive lipids, reducing non-specific adsorption in vivo. Adjusting the osmotic pressure to the isotonic range ensures the safety of the drug during application.

[0026] This invention provides a method for preparing smegglutinin using circRNA and related compositions. It has the following beneficial effects: 1. This invention constructs circRNA molecules with closed-loop structures and initiates translation using internal ribosome entry site sequences. By utilizing the lack of ends in the closed-loop structure, it avoids degradation by intracellular exonucleases, thereby prolonging the expression cycle of smegglutinin precursor protein. This improves the purity of the circRNA molecule stock solution and reduces the risk of immune responses caused by linear RNA impurities.

[0027] 2. This invention uses a lipid mixture comprising positive lipids, phospholipids, cholesterol, and polyethylene glycol lipids as a delivery carrier to encapsulate negatively charged circRNA molecules. The delivery efficiency is improved by utilizing the binding ability of positive lipids to RNA and the barrier effect of polyethylene glycol lipids. Furthermore, the addition of pharmaceutically acceptable excipients such as sugars and nonionic surfactants is used to prevent the lipid nanoparticles from fusing or aggregating during storage by utilizing the protective effect of sugars and the stabilizing effect of nonionic surfactants, thereby maintaining the physicochemical stability of the pharmaceutical composition.

[0028] 3. This invention utilizes a microfluidic chip system to control the mixing of an organic phase solution containing a lipid mixture with an aqueous phase solution containing circRNA molecules. By adjusting the total flow rate and volumetric flow rate ratio, lipid molecules are ordered to assemble and encapsulate circRNA molecules to obtain crude lipid nanoparticles with uniform particle size distribution. In conjunction with a subsequent dialysis step to remove residual organic solvents and replace the buffer system, and by adjusting the osmotic pressure and pH value of the solution to a physiologically suitable range, the final smegglutide circRNA drug composition is ensured to have good biocompatibility. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the circRNA molecule structure of the present invention; Figure 2 This is a map of the recombinant vector pUC57 of the present invention. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Preparation Examples 1-3: Preparation Example 1: This preparation example provides a circRNA molecular stock solution for preparing smegglutinin, including the following steps: Design and synthesize a DNA sequence containing the T7 promoter, the 5' end of the Anabaena intron, the encephalomyocarditis virus IRES (SEQ ID NO:5), the smegglutinin precursor ORF, and the 3' end of the Anabaena intron (its molecular structure diagram is shown in Figure 1). Figure 1 (As shown). The semaglutide precursor ORF encodes the complete semaglutide precursor protein (SEQ ID NO:4), which, from the N-terminus to the C-terminus, comprises: a signal peptide (SEQ ID NO:2), a linker peptide (SEQ ID NO:3), and the mature semaglutide peptide (SEQ ID NO:1). The nucleotide sequence of this ORF is optimized according to human codon preference (92% homology).

[0032] The above sequence was cloned into the pUC57 vector (as shown in the attached image). Figure 2 As shown in the figure, the recombinant plasmid was taken and linearized using XbaI restriction endonuclease at 36°C. The enzyme digestion reaction time was controlled to be 2 hours, and the linear DNA template was recovered and purified. Establish a 20 μL transcription reaction system, add 1 μg of the above linearized DNA template, T7 RNA polymerase and nucleotide mixture, and incubate the reaction system at 36℃ for 2 hours; After the reaction was completed, DNase I was added and incubated at 36°C for 15 minutes to digest and remove the DNA template. Then, the linear RNA intermediate was obtained by phenol / chloroform extraction. The linear RNA prepared above was resuspended in a circularization buffer with a GTP concentration of 1.5 mmol / L and a MgCl2 concentration of 10 mmol / L. The RNA solution was heated to 50°C and held at this temperature for 5 minutes to promote intron-mediated self-splicing circularization. The sample was subjected to dT20 packing material chromatography, the sample solution was collected, and then subjected to molecular sieve chromatography to obtain smegglutinin circRNA stock solution A.

[0033] Preparation Example 2: This preparation example provides a circRNA molecular stock solution for preparing smegglutinin, including the following steps: A DNA sequence containing the T7 promoter, the 5' end of the *Anabaena* intron, the encephalomycoplasma virus IRES (SEQ ID NO:5), the smegglutinin precursor ORF, and the 3' end of the *Anabaena* intron was designed and synthesized. The smegglutinin precursor ORF encodes the complete smegglutinin precursor protein (SEQ ID NO:4), which, from the N-terminus to the C-terminus, comprises a signal peptide (SEQ ID NO:2), a linker peptide (SEQ ID NO:3), and the mature smegglutinin peptide (SEQ ID NO:1). The nucleotide sequence of this ORF was optimized according to human codon bias (95% homology).

[0034] The above sequence was cloned into the pUC57 vector. The recombinant plasmid was linearized using XbaI restriction endonuclease at 38°C. The enzyme digestion reaction time was controlled to be 3 hours. The linear DNA template was recovered and purified. Establish a 25 μL transcription reaction system, add 7 μg of the above linearized DNA template, T7 RNA polymerase and nucleotide mixture, and incubate the reaction system at 37°C for 3 hours; After the reaction was completed, DNase I was added and incubated at 38°C for 20 minutes to digest and remove the DNA template. Then, the linear RNA intermediate was obtained by phenol / chloroform extraction. The linear RNA prepared above was resuspended in a circularization buffer with a GTP concentration of 2.0 mmol / L and a MgCl2 concentration of 20 mmol / L. The RNA solution was heated to 52.5 °C and held at this temperature for 15 minutes to promote intron-mediated self-splicing circularization. The sample was subjected to affinity chromatography and ion exchange chromatography to obtain smegglutinin circRNA stock solution B.

[0035] Preparation Example 3: This preparation example provides a circRNA molecular stock solution for preparing smegglutinin, including the following steps: A DNA sequence containing the T7 promoter, the 5' end of the *Anabaena* intron, the encephalomycoplasma virus IRES (SEQ ID NO:5), the smegglutinin precursor ORF, and the 3' end of the *Anabaena* intron was designed and synthesized. The smegglutinin precursor ORF encodes the complete smegglutinin precursor protein (SEQ ID NO:4), which, from the N-terminus to the C-terminus, comprises a signal peptide (SEQ ID NO:2), a linker peptide (SEQ ID NO:3), and the mature smegglutinin peptide (SEQ ID NO:1). The nucleotide sequence of this ORF was optimized according to human codon bias (98% homology).

[0036] The above sequence was cloned into the pUC57 vector. The recombinant plasmid was linearized using XbaI restriction endonuclease at 40°C. The enzyme digestion reaction time was controlled to be 4 hours. The linear DNA template was recovered and purified. Establish a 30 μL transcription reaction system, add 5 μg of the above linearized DNA template, T7 RNA polymerase and nucleotide mixture, and incubate the reaction system at a constant temperature of 40℃ for 4 hours; After the reaction was completed, DNase I was added and incubated at 40°C for 25 minutes to digest and remove the DNA template. Then, the linear RNA intermediate was obtained by phenol / chloroform extraction. The linear RNA prepared above was resuspended in a circularization buffer with a GTP concentration of 2.5 mmol / L and a MgCl2 concentration of 30 mmol / L. The RNA solution was heated to 55°C and held at this temperature for 25 minutes to promote intron-mediated self-splicing circularization. The cyclization reaction product was purified to remove uncirculated linear RNA precursors and splicing byproducts. After the treatment, the product was recovered by ethanol precipitation and reconstituted with nuclease-free water to obtain smegglutinin circRNA stock solution C.

[0037] Examples 1-3: Example 1: This embodiment provides a semaglutide circRNA pharmaceutical composition, comprising the following steps: S1. Weigh the lipid materials DLin-MC3-DMA, DSPC, cholesterol and PEG-DMG, and dissolve them in anhydrous ethanol at a molar ratio of 45:10.5:43.5:1. Stir until completely dissolved to prepare an organic phase solution with a total lipid concentration of 10 mmol / L. S2. Take the stock solution A of smegglutinin circRNA prepared in Preparation Example 1, add it to citrate buffer (100 mmol / L, pH 3.8), dilute and adjust the mass concentration of circRNA to 0.1 mg / mL, and use it as an aqueous phase solution. S3. Place the organic phase solution prepared in step S1 and the aqueous phase solution prepared in step S2 into the storage tube of the microfluidic chip system, set the volume flow ratio of the aqueous phase to the organic phase to be 2:1, control the total flow rate to be 16 mL / min, start the microfluidic program to mix, and collect the outflowing crude lipid nanoparticle solution. S4. The crude product solution collected in step S3 is placed into a dialysis bag with a molecular weight cutoff of 100kDa and dialyzed in 100 times the volume of phosphate buffer. The dialysis time is controlled at 10 hours, and the dialysis solution is changed 3 times during the period to remove ethanol and replace the buffer system. S5. Add sucrose to the dialyzed lipid nanoparticle solution to a final concentration of 5% (mass-volume ratio), add Tween 80 to a final concentration of 0.01% (volume ratio), adjust the system to a phosphate buffer environment with a concentration of 10 mmol / L and pH 6.8, and adjust the osmotic pressure of the solution to 280 mOsm / kg. Filter the solution through a 0.20 μm filter membrane for sterilization to obtain the smegglutinin circRNA drug composition.

[0038] Example 2: This embodiment provides a semaglutide circRNA pharmaceutical composition, comprising the following steps: S1. Weigh the lipid materials DLin-MC3-DMA, DSPC, cholesterol and PEG-DMG, and dissolve them in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5. Stir until completely dissolved to prepare an organic phase solution with a total lipid concentration of 13 mmol / L. S2. Take the stock solution B of smegglutinin circRNA prepared in Preparation Example 2, add it to citrate buffer (20 mmol / L, pH 4.0), dilute and adjust the mass concentration of circRNA to 0.2 mg / mL, and use it as an aqueous phase solution. S3. Place the organic phase solution prepared in step S1 and the aqueous phase solution prepared in step S2 into the storage tube of the microfluidic chip system, set the volume flow ratio of the aqueous phase to the organic phase to be 3:1, control the total flow rate to be 20 mL / min, start the microfluidic program to mix, and collect the outflowing crude lipid nanoparticle solution. S4. The crude product solution collected in step S3 is placed into a dialysis bag with a molecular weight cutoff of 100kDa and dialyzed in 125 times the volume of phosphate buffer. The dialysis time is controlled at 12 hours, and the dialysis solution is changed 4 times during the period to remove ethanol and replace the buffer system. S5. Add sucrose to the dialyzed lipid nanoparticle solution to a final concentration of 10% (mass-volume ratio), add Tween 80 to a final concentration of 0.05% (volume ratio), adjust the system to a phosphate buffer environment with a concentration of 15 mmol / L and pH 7.0, and adjust the osmotic pressure of the solution to 300 mOsm / kg. Filter the solution through a 0.22 μm filter membrane for sterilization to obtain the smegglutinin circRNA drug composition.

[0039] Example 3: This embodiment provides a semaglutide circRNA pharmaceutical composition, comprising the following steps: S1. Weigh the lipid materials DLin-MC3-DMA, DSPC, cholesterol and PEG-DMG, and dissolve them in anhydrous ethanol at a molar ratio of 55:9.5:33.5:2. Stir until completely dissolved to prepare an organic phase solution with a total lipid concentration of 16 mmol / L. S2. Take the stock solution C of smegglutinin circRNA prepared in Preparation Example 3, add it to citrate buffer (100 mmol / L, pH 4.2), dilute and adjust the mass concentration of circRNA to 0.3 mg / mL, and use it as an aqueous phase solution. S3. Place the organic phase solution prepared in step S1 and the aqueous phase solution prepared in step S2 into the storage tube of the microfluidic chip system, set the volume flow ratio of the aqueous phase to the organic phase to be 4:1, control the total flow rate to be 24 mL / min, start the microfluidic program to mix, and collect the outflowing crude lipid nanoparticle solution. S4. The crude product solution collected in step S3 is placed into a dialysis bag with a molecular weight cutoff of 100kDa and dialyzed in 150 times the volume of phosphate buffer. The dialysis time is controlled at 14 hours, and the dialysis solution is changed 5 times during the period to remove ethanol and replace the buffer system. S5. Add mannitol to the dialyzed lipid nanoparticle solution to bring the final concentration to 15% (mass-volume ratio), add Tween 80 to bring the final concentration to 0.1% (volume ratio), adjust the system to a phosphate buffer environment with a concentration of 20 mmol / L and pH 7.2, and adjust the osmotic pressure of the solution to 320 mOsm / kg. Filter the solution through a 0.24 μm filter membrane for sterilization to obtain the smegglutinin circRNA drug composition.

[0040] Comparative Examples 1-4: Comparative Example 1: Compared with Example 2, the difference is that in step S2, the smegglutinin circRNA stock solution B is replaced with a linear mRNA solution encoding the same amino acid sequence (after capping and tailing treatment), while the other parameters and steps are the same.

[0041] Comparative Example 2: Compared with Example 2, the difference is that in step S3, the flow rate parameter of the microfluidic chip system is adjusted so that the average particle size of the collected lipid nanoparticle crude product is 200 nm. All other parameters and steps are the same.

[0042] Comparative Example 3: Compared with Example 2, the difference is that in step S1, the molar ratio of lipid materials DLin-MC3-DMA, DSPC, cholesterol and PEG-DMG is adjusted so that the zeta potential of the final lipid nanoparticles is +10mV. All other parameters and steps are the same.

[0043] Comparative Example 4: Compared with Example 2, the difference is that in step S2, smegglutinin circRNA stock solution B is not added, and only an equal volume of citrate buffer (20 mmol / L, pH 4.0) is used as the aqueous phase solution to prepare empty carrier lipid nanoparticles without active ingredients. All other parameters and steps are the same.

[0044] Test Example 1-3: Test Example 1: Validation of In Vitro Expression Efficiency and Bioactivity Experimental description: This experiment aimed to evaluate the translational expression efficiency of the semaglutide circRNA pharmaceutical composition prepared in the examples in eukaryotic cells and to verify whether its expression product possessed biological activity consistent with the natural protein. The pharmaceutical composition prepared in Example 2 was selected as the experimental subject, and the large-particle-size lipid nanoparticles prepared in Comparative Example 2 were used as a control. Empty carrier lipid nanoparticles prepared in Comparative Example 4 were also used as a blank reference. The experiment covered three dimensions: protein expression kinetics detection, receptor affinity determination, and verification of insulin secretion function.

[0045] Experimental steps: For protein expression kinetics assays, HEK293 cells were revived and cultured. When the cell confluence reached approximately 75%, samples from Example 2, Comparative Example 2, and Comparative Example 4 were added, maintaining the same effective drug concentration or cell seeding density in each group. During culture, cell culture supernatant was aspirated at 24, 48, and 72 hours post-drug administration and centrifuged to remove cell debris. The protein concentration in the supernatant was measured using a specific ELISA kit targeting semaglutide precursor protein, and the protein expression level at each time point was calculated based on a standard curve.

[0046] Before receptor affinity assays, expression products from each group were collected after 72 hours and purified by nickel column affinity chromatography and gel filtration chromatography to obtain high-purity target proteins. CHO-K1 / GLP-1R cells stably overexpressing the GLP-1 receptor were resuscitated and seeded in 96-well plates. After cell adhesion, serially diluted purified protein solutions from each group were added for incubation, with FITC-labeled competitive ligands added. After incubation, cells were washed, and the fluorescence intensity on the cell surface was detected by flow cytometry or a fluorescence microplate reader. A dose-response curve was fitted using nonlinear regression analysis, and the half-maximal effective concentration (EC50) was calculated to assess the binding affinity of the expression products to the GLP-1 receptor.

[0047] To validate insulin secretion function, mouse insulinoma cells (MIN6) were used as a model. MIN6 cells were seeded in culture plates, pre-incubated, and then equilibrated with KRP buffer containing a low concentration of glucose. Subsequently, purified protein solutions of each group were added to a final concentration of 10 nmol / L to stimulate the cells, and cultured for another 2 hours. The supernatant was collected, and insulin levels were measured using a mouse insulin ELISA kit. The fold change in insulin secretion was calculated based on the secretion levels of untreated blank cells.

[0048] Experimental data: The protein expression concentrations and biological activity indicators of each group of samples at different time points are shown in the table below.

[0049] Table 1. Summary of in vitro expression kinetics and biological activity test data

[0050] Note: Comparative Example 2 is based on the traditional CHO recombinant protein process. The data here reflects its early expression within the same 72-hour culture period; "—" indicates no specific binding and EC50 cannot be calculated.

[0051] Experimental conclusion: Based on the analysis of the experimental data in Table 1, the present invention scheme represented by Example 2 shows advantages in terms of expression efficiency and bioactivity.

[0052] Regarding expression efficiency, the protein concentration in cells of Example 2 reached 278.4 mg / L 72 hours after transfection, significantly higher than the expression level (46.2 mg / L) of Comparative Example 2 at the same time period. This difference verifies the influence of vector particle size on delivery efficiency: the lipid nanoparticles of Comparative Example 2 had a particle size of 200 nm. The larger particle size hindered the endocytic uptake efficiency of cells, resulting in a reduction in the number of circRNA templates available for translation within the cells, ultimately reducing protein expression levels. In contrast, Example 2 controlled the particle size within an appropriate range, achieving efficient cell transfection and protein expression.

[0053] Regarding biological activity, the expression products of Example 2 and Comparative Example 2 showed no significant differences in receptor binding affinity (EC50 values ​​of 0.81 nmol / L and 0.79 nmol / L, respectively) and insulin secretion-promoting capacity (3.18-fold and 3.22-fold, respectively). This indicates that the polypeptide chain translated from the circRNA template can fold and modify correctly to form an active protein with its native conformation, and its biological function is not affected by the vector type or expression level.

[0054] Test Example 2: In vivo pharmacokinetics and long-term effect test Experimental description: This experiment aimed to evaluate the metabolic kinetics of the smegglutinin circRNA drug compositions prepared in different embodiments in mammals and to examine the long-term expression capacity of the drug. The drug compositions prepared in Examples 1, 2, and 3 were selected as the experimental group to investigate the effect of the process parameter range of the present invention on the in vivo efficacy; the linear mRNA formulation prepared in Comparative Example 1 was selected as the structural control group; and the large-particle-size and positively charged lipid nanoparticles prepared in Comparative Example 3 were selected as the formulation parameter control group.

[0055] Experimental steps: SPF-grade male C57BL / 6 mice, aged 6 to 8 weeks, were randomly divided into 5 groups of 6 mice each. Mice were acclimatized for 3 days prior to the experiment with free access to food and water. On the day of drug administration, all mice in each group received a single intravenous injection via tail vein. The dosage was uniformly set at 1 mg / kg based on nucleic acid mass.

[0056] Following drug administration, blood samples were collected from the orbital venous plexus of mice on days 1, 3, 7, 14, and 21. After incubation at room temperature for 30 minutes, the collected whole blood samples were centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant serum was separated. The drug concentration in the serum was detected using a semaglutide-specific ELISA kit. During the assay, each sample was tested in replicates, and the serum drug concentration was calculated based on a standard curve, with data recorded.

[0057] Experimental data: The serum semaglutide concentration monitoring results of mice in each group at different time points are shown in the table below.

[0058] Table 2. Summary of serum semaglutide pharmacokinetic data in mice after a single dose.

[0059] Note: Not detected means the detection value is below the minimum detection limit of the ELISA kit.

[0060] Experimental conclusion: The pharmacokinetic data in Table 2 show that the drug compositions prepared in Examples 1 to 3 have good long-term expression characteristics in vivo.

[0061] In the example group, mice reached peak blood drug concentration on day 3 after drug administration, maintained a concentration of approximately 10 ng / mL on day 14, and still had drug residues on day 21. This confirms that the circRNA prepared in this invention, due to its closed circular structure, can reduce degradation by endogenous ribonucleases and achieve sustained translation in vivo for a longer period. In contrast, the linear mRNA in Comparative Example 1 showed a significant decrease in concentration on day 3 and was undetectable on day 7, indicating that the circular structure plays an important role in maintaining blood drug concentration in vivo.

[0062] Meanwhile, the example group controlled the particle size of the lipid nanoparticles to be between 50 and 100 nm and had a slightly negatively charged surface. This parameter range is beneficial for drug delivery in vivo. Compared with Comparative Example 3, the example group showed a higher peak plasma drug concentration and a longer duration of action, indicating that suitable particle size and potential help the nanoparticles remain in the bloodstream and be taken up by target tissues, thereby improving drug bioavailability. In summary, the pharmaceutical composition of the present invention achieves long-term expression after a single dose by optimizing the nucleic acid structure and carrier parameters.

[0063] Test Example 3: In vivo pharmacodynamic testing – Oral glucose tolerance test Experimental description: This experiment aimed to evaluate the biological effects of the smegglutinin circRNA pharmaceutical composition prepared in the examples in vivo, specifically by examining its ability to regulate glycemic homeostasis through an oral glucose tolerance test. To verify the long-term mechanism of action, mice were selected for testing on day 7 after administration. The pharmaceutical composition prepared in Example 2 was selected as the experimental group, and the empty carrier lipid nanoparticles without active ingredients prepared in Comparative Example 4 were selected as the negative control group.

[0064] Experimental steps: Mice in the experimental group and negative control group, who had completed prior drug treatment and were on day 7 post-treatment, were selected. All mice were fasted for 12 hours before testing, but were allowed free access to water. After fasting, fasting blood glucose levels were measured in each group as the baseline at 0 minutes. Subsequently, mice were administered glucose solution by gavage at a dose of 2 g / kg body weight.

[0065] After gavage, timing began, and blood samples were collected via tail vein puncture at 30, 60, and 120 minutes. Whole blood glucose concentrations were measured at each time point using a calibrated medical-grade blood glucose meter. All raw data were recorded, the mean blood glucose level and standard deviation for each group at different time points were calculated, and a curve of blood glucose versus time was plotted to assess glucose tolerance.

[0066] Experimental data: The results of monitoring the dynamic changes in blood glucose levels in each group of mice after oral glucose loading are shown in the table below.

[0067] Table 3. Blood glucose monitoring data from the oral glucose tolerance test on day 7 of drug administration in mice.

[0068] Experimental conclusion: According to the pharmacodynamic data in Table 3, the pharmaceutical composition prepared in Example 2 still showed significant hypoglycemic activity on day 7 after administration.

[0069] Thirty minutes after oral glucose loading, at the peak blood glucose level, the blood glucose level in the experimental group mice was 10.18 mmol / L, significantly lower than the 15.86 mmol / L in the negative control group. This indicates that the semaglutide molecules expressed in vivo can effectively respond to the blood glucose elevation signal and inhibit postprandial blood glucose rise. By 120 minutes, the blood glucose level in the experimental group mice had dropped to 4.41 mmol / L, essentially returning to the fasting baseline level, while the negative control group remained at a higher level. This further confirms the drug's ability to regulate blood glucose homeostasis.

[0070] The test results were obtained on day 7 after a single dose, which is consistent with the pharmacokinetic data in Test Example 2. This indicates that the circRNA prepared in this invention produces an active smegglutinin through continuous translation in vivo, which can maintain its efficacy for a longer period of time and achieve long-term regulation of blood glucose.

[0071] Appendix: Mature peptide amino acid sequence of Smeglucopyranoside: SEQ ID NO:1: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.

[0072] Signal peptide amino acid sequence: SEQ ID NO:2: MKSIYFVAGLFVMLVQGSWQ.

[0073] Linking peptide amino acid sequence: SEQ ID NO:3: GRKRR.

[0074] Smegglutinin precursor protein amino acid sequence: SEQ ID NO:4: MKSIYFVAGLFVMLVQGSWQGRKRRHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.

[0075] Encephalomyocarditis virus nucleotide sequence: SEQ ID NO:5: CCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACC。

Claims

1. A circRNA molecule for use in the preparation of semaglutide peptide, characterized in that, The circRNA molecule comprises an open reading frame encoding a semaglutide precursor protein and an internal ribosome entry site sequence; The internal ribosome entry site sequence is selected from the encephalomyocarditis virus IRES, and the nucleotide sequence of the encephalomyocarditis virus IRES is shown as SEQ ID NO: 5; The amino acid sequence of the semaglutide precursor protein is shown as SEQ ID NO: 4, and the semaglutide precursor protein comprises, in sequence from N-terminus to C-terminus: a signal peptide, the amino acid sequence of which is shown as SEQ ID NO: 2; a linker peptide, the amino acid sequence of which is shown as SEQ ID NO: 3; a semaglutide mature peptide, the amino acid sequence of which is shown as SEQ ID NO:

1.

2. The circRNA molecule for preparing semaglutide peptide according to claim 1, characterized in that, The circRNA molecule is prepared by the following steps: constructing a recombinant plasmid containing a T7 promoter, a fishy algae intron 5' end, the internal ribosome entry site sequence, the semaglutide precursor protein coding sequence, and a fishy algae intron 3' end, recovering after linearization treatment by a restriction enzyme, to obtain a linear DNA template; performing an in vitro transcription reaction by the linear DNA template to obtain a linear RNA intermediate; performing a self-splicing circularization reaction on the linear RNA intermediate in a circularization buffer containing GTP and MgCl2 to obtain a circularization reaction product; purifying to remove uncircularized linear RNA precursors and splicing by-products, and obtaining a circRNA molecule stock solution after purification.

3. The circRNA molecule for the preparation of semaglutide according to claim 2, characterized in that, The linearization treatment by the restriction enzyme is performed at 36-40°C using XbaI restriction enzyme, and the treatment time is 2-4 hours; The control reaction temperature of the in vitro transcription reaction is 36-40°C, and the reaction time is 2-4 hours.

4. The circRNA molecule for preparing semaglutide peptide according to claim 2, characterized in that, The concentration of GTP in the circularization buffer is 1.5-2.5 mmol / L, and the concentration of MgCl2 is 10-30 mmol / L; The control reaction temperature of the self-splicing circularization reaction is 50-55°C, and the reaction time is 5-25 minutes.

5. A semaglutide circRNA pharmaceutical composition, characterized in that, The raw materials comprise: The circRNA molecule of claim 1; a lipid mixture consisting of, in terms of molar fraction, 45-55 parts of positive lipids, 9.5-10.5 parts of phospholipids, 33.5-43.5 parts of cholesterol, and 1-2 parts of polyethylene glycol lipids; an auxiliary material comprising a sugar and a non-ionic surfactant, wherein the final concentration of the sugar is 5% (g / v) to 15% (g / v), and the final concentration of the non-ionic surfactant is 0.01% (v / v) to 0.1% (v / v).

6. The semaglutide circRNA pharmaceutical composition according to claim 5, wherein The positive lipids are DLin-MC3-DMA; the phospholipids are DSPC; the polyethylene glycol lipids are PEG-DMG; the sugar is selected from sucrose or mannitol; and the non-ionic surfactant is Tween 80.

7. A method of preparing a semaglutide circRNA pharmaceutical composition, characterized in that, A semaglutide circRNA pharmaceutical composition as claimed in any one of claims 5-6 is prepared by the following steps: dissolving the lipid mixture in an organic solvent to obtain an organic phase solution; dissolving the circRNA molecule in an acidic buffer to obtain an aqueous phase solution; mixing the organic phase solution and the aqueous phase solution by using a microfluidic chip system, collecting the effluent to obtain a crude lipid nanoparticle preparation; placing the crude lipid nanoparticle preparation in a dialysis medium to dialyze, remove the organic solvent and replace the buffer system, to obtain a dialyzed lipid nanoparticle solution; adding excipients to the dialyzed lipid nanoparticle solution to adjust the osmotic pressure and pH value of the solution, and filtering to obtain the semaglutide circRNA pharmaceutical composition.

8. The method of claim 7, wherein the semaglutide circRNA pharmaceutical composition is prepared by the steps of: During the solution preparation process: the total lipid concentration of the organic phase solution is 10 mmol / L to 16 mmol / L; the mass concentration of the aqueous phase solution is 0.1 mg / mL to 0.3 mg / mL; the acidic buffer is a citric acid buffer with a pH of 3.8 to 4.

2.

9. The method of claim 7, wherein the semaglutide circRNA pharmaceutical composition is prepared by the steps of: During the mixing process: the total flow rate of the microfluidic chip system during mixing is controlled to be 16 mL / min to 24 mL / min; the volume flow ratio of the aqueous phase solution to the organic phase solution is controlled to be 2:1 to 4:

1.

10. The method of claim 7, wherein the semaglutide circRNA pharmaceutical composition is prepared by the steps of: During the dialysis and adjustment process: the volume of the dialysis medium is 100 times to 150 times the volume of the crude lipid nanoparticle preparation, and the dialysis time is 10 hours to 14 hours; the buffer system is a phosphate buffer with a pH of 6.8 to 7.2; the osmotic pressure of the adjusted semaglutide circRNA pharmaceutical composition solution is 280 mOsm / kg to 320 mOsm / kg.

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