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KR-20260067385-A - Fusion polypeptide, expression cassette containing the coding gene of the fusion polypeptide, gene transfer vector, pharmaceutical composition and use

KR20260067385AKR 20260067385 AKR20260067385 AKR 20260067385AKR-20260067385-A

Abstract

The present invention provides a fusion polypeptide, an expression cassette containing the coding gene thereof, a gene delivery vector, a pharmaceutical composition, and uses. Specifically, the invention provides a fusion polypeptide comprising a secretion signal peptide and an aflibercept polypeptide, wherein the secretion signal peptide is selected from any one of a human albumin secretion signal peptide, a human Opticin protein secretion signal peptide, a human interleukin-2 secretion signal peptide, and a mouse receptor tyrosine kinase-like orphan receptor 1 secretion signal peptide. The provided fusion polypeptide, a polynucleotide encoding the same, a gene expression cassette containing the polynucleotide, and a gene delivery vector containing the expression cassette can enhance the expression of aflibercept and improve the efficacy of treating eye diseases using aflibercept.

Inventors

  • 왕, 쳉

Assignees

  • 이노벡 바이오테라퓨틱스

Dates

Publication Date
20260512
Application Date
20240725
Priority Date
20230801

Claims (17)

  1. A fusion polypeptide comprising a secretory signal peptide and an aflibercept polypeptide, wherein the secretory signal peptide is selected from any one of a human albumin secretory signal peptide, a human Opticin protein secretory signal peptide, a human interleukin-2 secretory signal peptide, and a mouse receptor tyrosine kinase-like orphan receptor 1 secretory signal peptide.
  2. In claim 1, The amino acid sequence of the aflibercept polypeptide described above comprises the amino acid sequence indicated in SEQ ID NO: 48, and/or A fusion polypeptide in which the amino acid sequence of the above secretion signal peptide comprises the amino acid sequence indicated in any one of SEQ ID NO: 44 to 47.
  3. In claim 1 or 2, A fusion polypeptide in which the amino acid sequence of the above fusion polypeptide comprises any one of the following sequences. (i) Amino acid sequence indicated in any one of SEQ ID NO: 1, 30, 32 or 34, (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence indicated in any one of SEQ ID NO: 1, 30, 32, or 34, wherein the amino acid sequence retains the function of the amino acid sequence indicated in any one of SEQ ID NO: 1, 30, 32, or 34, (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted in the amino acid sequence indicated by any one of SEQ ID NO: 1, 30, 32, or 34, and which retains the function of the amino acid sequence indicated by any one of SEQ ID NO: 1, 30, 32, or 34, or (iv) An amino acid sequence encoded by a nucleotide sequence that hybridizes under strict conditions with a polynucleotide sequence encoding an amino acid sequence indicated in any one of SEQ ID NO: 1, 30, 32 or 34, wherein said amino acid sequence retains the function of the amino acid sequence indicated in any one of SEQ ID NO: 1, 30, 32 or 34, and said strict conditions are moderately strict conditions, moderate to high strict conditions, high strict conditions, or ultra-high strict conditions.
  4. As an isolated polynucleotide encoding a fusion polypeptide described in any one of claims 1 to 3, Preferably, the polynucleotide comprises an isolated polynucleotide having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence indicated in any one of SEQ ID NO: 2, 29, 31, or 33.
  5. A gene expression cassette comprising the polynucleotide described in claim 4.
  6. In claim 5, A gene expression cassette comprising one or more of a promoter, enhancer, intron, and Kojak sequence upstream of a polynucleotide encoding a fusion polypeptide.
  7. In claim 6, The above promoter includes any one of the CBh promoter, the CB7 promoter, and the CMV promoter, and Preferably, the CBh promoter comprises the sequence indicated in SEQ ID NO: 3, or a sequence having at least 85% identity with it, and/or The above CB7 promoter comprises the sequence indicated in SEQ ID NO: 4, or a sequence having at least 85% identity with it, and/or The above CMV promoter comprises a sequence indicated in SEQ ID NO: 5, or a sequence having at least 85% identity with it, in a gene expression cassette.
  8. In claim 6 or 7, The above intron includes human β-globulin introns, and Preferably, the human β-globulin intron comprises the sequence indicated in SEQ ID NO: 6, or a sequence having at least 85% identity with it, in a gene expression cassette.
  9. In any one of claims 5 to 8, The polynucleotide encoding the fusion polypeptide includes a polyadenylation region downstream of the polynucleotide, and Optionally, the polyadenylation region is any one selected from bovine growth hormone, human growth hormone, and the polyadenylation region of β-globin, and Preferably, the polyadenylated region comprises a human growth hormone polyadenylated region, and More preferably, the human growth hormone polyadenylation region comprises the sequence indicated in SEQ ID NO: 7, or a sequence having at least 85% identity with it, in a gene expression cassette.
  10. In any one of claims 5 to 9, A gene expression cassette comprising a sequence indicated in any one of SEQ ID NO: 8 to 10, 35 to 43, or a sequence having at least 85% identity with any one of SEQ ID NO: 8 to 10, 35 to 43.
  11. A gene transfer vector comprising a gene expression cassette as described in any one of claims 5 to 10.
  12. In claim 11, The above gene transfer vector is a virus-derived viral vector, and Preferably, the gene transfer vector is a recombinant adeno-associated virus.
  13. In claim 12, The above-mentioned recombinant adeno-associated virus includes a capsid protein, and the gene expression cassette is capsidized within the capsid protein, and Optionally, the above-mentioned capsid protein is selected from any one of adeno-associated virus serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10, or from a variant thereof, in a gene transfer vector.
  14. In claim 13, The above capsid protein is an AAV2 capsid protein or a variant thereof, and Preferably, the capsid protein is an AAV2 capsid protein variant, and More preferably, the AAV2 capsid protein variant comprises a sequence indicated in any one of SEQ ID NO: 16, 70 to 83, or a sequence having at least 85% identity with any one of SEQ ID NO: 16, 70 to 83, and More preferably, the AAV2 capsid protein variant comprises a sequence indicated in SEQ ID NO: 16 or 82, or a sequence having at least 85% identity with SEQ ID NO: 16 or 82, a gene transfer vector.
  15. A fusion polypeptide as described in any one of claims 1 to 3, a polynucleotide as described in claim 4, a gene expression cassette as described in any one of claims 5 to 10, or a gene transfer vector as described in any one of claims 11 to 14, A pharmaceutical composition comprising, in some cases, a pharmaceutically acceptable carrier.
  16. In the manufacture of a medicine for the prevention and/or treatment of a disease, by using a fusion polypeptide described in any one of claims 1 to 3, a polynucleotide described in claim 4, a gene expression cassette described in any one of claims 5 to 10, or a gene transfer vector described in any one of claims 11 to 14, The above disease is arbitrarily an eye disease, and Preferably, the eye disease is associated with neovascularization of the eye or with neovascularization of the choroid, and More preferably, the eye disease is selected from one or more of exudative age-related macular degeneration, macular edema associated with retinal vein occlusion, diabetic macular edema, and diabetic retinopathy.
  17. A method for the prevention and/or treatment of a disease comprising administering to a subject an effective amount of a fusion polypeptide described in any one of claims 1 to 3, a polynucleotide described in claim 4, a gene expression cassette described in any one of claims 5 to 10, a gene transfer vector described in any one of claims 11 to 14, or a pharmaceutical composition described in claim 15, The above disease is arbitrarily an eye disease, and Preferably, the eye disease is associated with neovascularization of the eye or with neovascularization of the choroid, and More preferably, the eye disease is selected from one or more of exudative age-related macular degeneration, macular edema associated with retinal vein occlusion, diabetic macular edema, and diabetic retinopathy, a method for preventing and/or treating the disease.

Description

Fusion polypeptide, expression cassette containing the coding gene, gene transfer vector, pharmaceutical composition and use (Priority and related applications) The present disclosure claims priority to PCT international application PCT/CN2023/110563, filed on August 1, 2023, with the title of the invention "fusion polypeptide, expression cassette containing the coding gene thereof, gene transfer vector, pharmaceutical composition and use," the entire contents of said application, including the appendix, are incorporated into the present disclosure by reference. (Technology field) The present disclosure belongs to the field of gene therapy technology, and specifically relates to a fusion polypeptide, an expression cassette containing the coding gene, a gene delivery vector, a pharmaceutical composition, and uses. Pathological neovascularization-related eye diseases, such as Wet Age-related Macular Degeneration (Wet AMD), have a rapid onset and lead to irreversible, severe vision loss or blindness within a short period. Although Wet AMD accounts for only 20% of all AMD cases, the blindness rate reaches 80%, significantly impacting the quality of life for patients and their families. Wet AMD is the third most common cause of blindness globally and is a major cause of vision loss in the elderly. The incidence rate in China is also increasing annually, with approximately 9 million people currently suffering from Wet AMD. Among adults over 50 in China, the proportion of AMD patients is 15.5%, and Wet AMD accounts for 11.9% of all AMD cases. Currently, there is no effective treatment for Wet AMD. The mainstream treatment is anti-angiogenic therapy. This method primarily involves regularly injecting recombinant anti-VEGF proteins or antibodies into the vitreous humor. However, this traditional treatment has significant drawbacks. The need for repeated administration has serious adverse effects on patients, leading to poor medication adherence and severe side effects. Therefore, developing a gene therapy capable of treating this disease in a single treatment is a very urgent task. Aflibercept is a recombinant fusion protein that functions as a decoy receptor for vascular endothelial growth factor subtypes A and B (VEGF-A and VEGF-B) and placental growth factor (PlGF). By binding to these ligands, Aflibercept inhibits the binding of these ligands to the vascular endothelial growth factor receptors (VEGFR), namely VEGFR-1 and VEGFR-2, thereby inhibiting angiogenesis and potentially reducing vascular permeability. Aflibercept consists of Domain 2 of VEGFR-1 and Domain 3 of VEGFR-2 fused to the Fc region of IgG1. Aflibercept is an ophthalmic intravitreal Aflibercept fusion protein injection, product name It is being marketed as (aflibercept). Recently, gene therapy has been becoming increasingly mature and safe through further improvements in gene introduction and delivery efficiency, as well as significant advancements in clinical research. In 2008, gene therapy garnered renewed attention due to the successful treatment of Leber's congenital amaurosis (LCA) using an adeno-associated virus vector. In 2012, Glybera, an adeno-associated virus-derived gene therapy, opened a new era of gene therapy when it received its first approval from the European Medicines Agency (EMA). In 2017, the FDA approved Luxturna, a gene therapy using Adeno-Associated Virus 2 (AAV2), an adeno-associated virus vector carrying the RPE65 gene, as a treatment for vision loss caused by Inherited Retinal Diseases (IRDs). Since then, the field of gene therapy has advanced rapidly, with clinical cases and related drugs being reported in succession, and the safety and efficacy of gene therapy gradually gaining recognition. AAV viral vectors have become a major application model in gene therapy, and one of the important directions for gene therapy via recombinant AAV delivery is the central nervous system (CNS), including the eyes and brain. The eye is a relatively closed environment, and gene therapy delivered by directly injecting AAV into the eye can achieve therapeutic effects for various hereditary eye diseases. Clinical trials have shown that gene therapy via intraocular AAV delivery does not cause systemic side effects or induce serious immune responses. Figure 1a is a schematic diagram of the pssAAV-CMV-Fluc-hGH pA vector structure. ITR: Inverted terminal repeat (the length of the upstream ITR is approximately 145 bp, and the length of the downstream ITR is approximately 141 bp). CMV promoter: The early promoter of the human cytomegalovirus. Human beta Globin intron: Human β-globulin intron. Fluc: The coding sequence of the firefly luciferase gene. hGH polyA: The polynucleotide tailing signal of human growth hormone. Kan: The leading frame of the kanamycin resistance gene. Figure 1b is a schematic diagram of the pssAAV-CMV-Aflibercept vector structure. ITR: Inverted terminal repeat (the length of the upstream ITR is approximately 145 bp, and the