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CN-121991240-A - Glucagon fusion proteins, polynucleotides and uses

CN121991240ACN 121991240 ACN121991240 ACN 121991240ACN-121991240-A

Abstract

The invention provides glucagon fusion protein, polynucleotide and application, and relates to the field of biotechnology. The glucagon fusion protein comprises a structure shown in a formula (I), wherein [ GCG ] - [ (L) - (GCG) ] m -[X] n , GCG is a glucagon fragment, L is a connecting peptide, X is a fragment with an amino acid sequence shown as SEQ ID No.2, m is an integer more than or equal to 0, n is 0 or 1, and m and n are not simultaneously 0. The glucagon fusion protein successfully solves the problem that glucagon cannot be expressed normally on the protein level by adding carbon end extension or short peptide concatemer, and provides a new idea for effectively regulating and controlling related metabolic pathways.

Inventors

  • XU MIN
  • Pei ya

Assignees

  • 派格生物医药(杭州)股份有限公司

Dates

Publication Date
20260508
Application Date
20241031

Claims (10)

  1. 1. A glucagon fusion protein, which comprises a polypeptide sequence, the device is characterized by comprising the following structures: [ GCG ] - [ (L) - (GCG) ] m -[X] n ; GCG is a glucagon fragment, L is a connecting peptide; x is a fragment with an amino acid sequence shown as SEQ ID No. 2; m is an integer not less than 0, n is 0 or 1, and m and n are not simultaneously 0.
  2. 2. The glucagon fusion protein of claim 1, wherein the amino acid sequence of each GCG is independently set forth in SEQ ID No. 1; Optionally, the connecting peptide is a flexible connecting peptide; optionally, the connecting peptide is (GGGGS) x , and x is a positive integer greater than or equal to 1; Optionally, the glucagon fusion protein comprises the structure: formula (i) [ GCG ] - [ X ], or, Formula (ii) [ GCG ] - [ (L) - (GCG) ], or, Formula (iii) [ GCG ] - [ (L) - (GCG) ] - [ X ]; Alternatively, the amino acid sequence of the glucagon fusion protein comprises the sequence shown as SEQ ID No.39, 40 or 41.
  3. 3. A polynucleotide comprising a CDS encoding a structure of formula (II) or formula (III): the formula (II) is [ Y ] - [ GCG ] - [ (L1) - (GCG) ] m -[X] n , or, The formula (III) is [ Y ] - [ GCG ] - [ (L2) - (Y) - (GCG) ] m -[X] n ; Y is a signal peptide, GCG is a glucagon fragment, L1 is a flexible connecting peptide, L2 is a cleavable connecting peptide, and X is a fragment with an amino acid sequence shown as SEQ ID No. 2; m is an integer more than or equal to 0, n is 0 or 1, and m and n are not simultaneously 0; Alternatively, n in formula (III) is 0; Optionally, the linker peptide L1 is (GGGGS) x , and x is a positive integer greater than or equal to 1; Alternatively, the signal peptide Y is a preproinsulin signal peptide; Optionally, the amino acid sequence of the preproinsulin signal peptide is shown in SEQ ID No. 3.
  4. 4. A polynucleotide according to claim 3, wherein the polynucleotide comprises DNA; alternatively, the DNA sequence encoding GCG is shown in SEQ ID NO.24 or 25; Alternatively, the DNA sequence encoding the preproinsulin signal peptide is shown in SEQ ID NO. 26; alternatively, the DNA sequence encoding the connecting peptide L1 is shown in SEQ ID NO. 27; Alternatively, the DNA sequence encoding X is shown as SEQ ID NO. 28; Alternatively, the polynucleotide is DNA, and the nucleotide sequence is shown as SEQ ID NO.21 or 23.
  5. 5. A polynucleotide according to claim 3, wherein the polynucleotide comprises RNA; alternatively, the RNA sequence encoding GCG is shown in SEQ ID NO.29 or 30; alternatively, the RNA sequence encoding the preproinsulin signal peptide is shown in SEQ ID NO. 31; alternatively, the RNA sequence encoding the connecting peptide L1 is shown in SEQ ID NO. 32; Alternatively, the RNA sequence encoding X is shown as SEQ ID NO. 33; Alternatively, the RNA comprises mRNA or circular RNA; alternatively, the nucleotide sequence of CDS of mRNA or circular RNA is shown as SEQ ID NO.15 or 18 respectively and independently; Alternatively, the structure of the mRNA includes 5'UTR-Kozak-CDS-3' UTR-polyA; alternatively, the nucleotide sequence of the 5' UTR is shown as SEQ ID NO. 34; Alternatively, kozak has a nucleotide sequence GCCACC; Alternatively, the nucleotide sequence of the 3' UTR is shown as SEQ ID NO. 36; Alternatively, the nucleotide sequence of polyA is shown as SEQ ID NO. 35; alternatively, the structure of the circular RNA includes E2 exon-5 'spacer-internal ribosome entry site-Kozak-CDS-3' spacer-E1 exon; alternatively, the nucleotide sequence of the E2 exon is shown as SEQ ID NO. 19; Alternatively, the nucleotide sequence of the 5' spacer is shown as SEQ ID NO. 37; Alternatively, the nucleotide sequence of the internal ribosome entry site is shown in SEQ ID NO. 14; alternatively, the nucleotide sequence of the 3' spacer is shown as SEQ ID NO. 38; alternatively, the nucleotide sequence of the E1 exon is shown as SEQ ID NO. 20.
  6. 6. A vector carrying the polynucleotide of any one of claims 3 to 5.
  7. 7. A cell expressing the glucagon fusion protein of claim 1 or 2, or carrying the polynucleotide of any one of claims 3 to 5, or comprising the vector of claim 6.
  8. 8. A method of preparing a glucagon fusion protein comprising expressing the glucagon fusion protein in a cell; optionally, the preparation method comprises introducing the polynucleotide of any one of claims 3 to 5 into a cell; optionally, the polynucleotide is mRNA, and the preparation method further comprises preparing mRNA by in vitro transcription; optionally, the polynucleotide is a circular RNA, and the method of making further comprises forming the circular RNA by a type I intron.
  9. 9. Use of a glucagon fusion protein of claim 1 or 2, or a polynucleotide of any one of claims 3-5, or a vector of claim 6, or a cell of claim 7, or a method of preparation of claim 8, for the preparation of a pharmaceutical composition for the prevention and/or treatment of hypoglycemia. The pharmaceutical composition is used for preventing and/or treating metabolic disorders; Alternatively, the metabolic disorder comprises hypoglycemia, type I diabetes, kidney disease caused by a metabolic disorder, fatty liver, or obesity.
  10. 10. A pharmaceutical composition comprising the glucagon fusion protein of claim 1 or 2, or the polynucleotide of any one of claims 3 to 5, or the vector of claim 6, or the cell of claim 7, for use in the prevention and/or treatment of metabolic disorders; Optionally, the metabolic disorder comprises hypoglycemia, type I diabetes, kidney disease caused by a metabolic disorder, fatty liver, or obesity; Alternatively, the polynucleotide comprises mRNA or circular RNA; alternatively, the nucleotide sequence of CDS of mRNA or circular RNA is shown as SEQ ID NO.15 or 18 respectively and independently; optionally, the pharmaceutical composition further comprises a GLP-1 receptor agonist and/or a GIP receptor agonist.

Description

Glucagon fusion proteins, polynucleotides and uses Technical Field The invention relates to the field of biotechnology, in particular to glucagon fusion protein, polynucleotide and application. Background The following statements merely provide background information related to the present disclosure and may not necessarily constitute prior art. Glucagon (GCG) is a peptide hormone secreted by islet a cells, regulating blood glucose levels and lipid metabolism. GCG plays a critical role in maintaining glucose homeostasis in animals and humans as an antagonistic hormone to insulin. To increase blood glucose, GCG acts synergistically through a variety of mechanisms to promote hepatic glucose output, increase glycogenosis and gluconeogenesis, and coordinate glycemic production by reducing glycogen synthesis and glycolysis. In contrast to healthy subjects, diabetics and animals have abnormal insulin secretion, but also GCG secretion. Hyperinsulinemia and changes in the ratio of insulin to GCG play a critical role in the initiation and maintenance of pathological hyperglycemic states. Therefore, GCG and GCG receptors have been widely pursued in recent years as potential targets for diabetes treatment. Currently, GCGR agonists are often used in combination with GLP-1R agonists, often directly for treatment in polypeptide form, but they suffer from a short half-life. Considering that RNA has higher expression level and longer half-life, we explored a new approach, RNA-mediated protein replacement therapy. However, GCG is a short peptide consisting of 29 amino acids, whose coding sequence has a low molecular weight, which makes its expression at the protein level challenging. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a glucagon fusion protein and a polynucleotide thereof, which are used for solving the problem that glucagon is difficult to express. Based on the glucagon fusion protein, the invention also provides a vector, a cell and a preparation method related to the glucagon fusion protein and application of the glucagon fusion protein. In order to solve the technical problems, the invention adopts the following technical scheme: in a first aspect, there is provided a glucagon fusion protein comprising the structure: [ GCG ] - [ (L) - (GCG) ] m-[X]n; GCG is a glucagon fragment, L is a connecting peptide; x is a fragment with an amino acid sequence shown as SEQ ID No. 2; m is an integer not less than 0, n is 0 or 1, and m and n are not simultaneously 0. In a second aspect, there is also provided a polynucleotide comprising a CDS encoding a structure of formula (II) or formula (III): the formula (II) is [ Y ] - [ GCG ] - [ (L1) - (GCG) ] m-[X]n, or, The formula (III) is [ Y ] - [ GCG ] - [ (L2) - (Y) - (GCG) ] m-[X]n; Y is a signal peptide, GCG is a glucagon fragment, L1 is a flexible connecting peptide, L2 is a cleavable connecting peptide, and X is a fragment with an amino acid sequence shown as SEQ ID No. 2; m is an integer not less than 0, n is 0 or 1, and m and n are not simultaneously 0. In a third aspect, there is also provided a vector carrying the polynucleotide of the second aspect. In a fourth aspect, there is also provided a cell which is a glucagon fusion protein according to the first aspect, or which carries a polynucleotide according to the second aspect, or which comprises a vector according to the third aspect. In a fifth aspect, there is also provided a method of preparing a glucagon fusion protein comprising expressing the glucagon fusion protein in a cell. In a sixth aspect there is also provided the use of a glucagon fusion protein of the first aspect, or a polynucleotide of the second aspect, or a vector of the third aspect, or a cell of the fourth aspect, or a method of manufacture of the fifth aspect, in the manufacture of a medicament for the prevention and/or treatment of a metabolic disorder. In a seventh aspect, there is also provided a medicament comprising a glucagon fusion protein of the first aspect, or a polynucleotide of the second aspect, or a vector of the third aspect, or a cell of the fourth aspect, for use in the prevention and/or treatment of a metabolic disorder. Compared with the prior art, the invention has the following beneficial effects: The invention successfully overcomes the problem that wild glucagon is difficult to express by adding signal peptide, carbon end extension or short peptide concatemer, relieves the limit that the biological fermentation method can not lead the glucagon to be expressed normally on the protein level, and provides a new idea for more effectively regulating and controlling related metabolic pathways. In preferred embodiments, the glucagon analogs are encoded by mRNA and circRNA, overcoming limitations of conventional recombinant protein therapies, such as short half-life, instability and immunogenicity of the protein, and the inability to replace intracellular proteins. The