Transporter peptides and uses thereof

Abstract

The present invention relates to a transporter peptide capable of binding to the transferrin receptor. The transporter peptide is conjugated to an effector in a covalent or non-covalent manner to form a transporter peptide conjugate, or the transporter peptide and effector form a recombinant transporter peptide conjugate by expressing nucleic acids encoding the transporter peptide and the effector. The transporter peptide and the recombinant transporter peptide transport the effector to a target by binding to the transferrin receptor. Binding of the transporter peptide to the transferrin receptor on cells at a tissue barrier induces the transcytosheath of the cells, thereby transporting the transporter peptide conjugate across the tissue barrier. The transporter peptide can be used as a drug delivery system for the treatment of central nervous system (CNS) diseases.

Description

[Technical Field]

[0001] The present invention relates to transporter peptides that span tissue barriers and methods of using these transporter peptides to transport effectors across tissue barriers. [Background technology]

[0002] The main function of tissue barriers is to restrict the passage of substances. Tissue barriers exist in various tissues and organs in living organisms to maintain the environmental stability of a given area or to protect important organs from the effects of external substances, and examples of such barriers include the blood-brain barrier (BBB) ​​and the gastrointestinal mucosal barrier.

[0003] The blood-brain barrier (BBB) ​​is located in the central nervous system and consists of cerebral microvascular endothelial cells (BMECs). Its main function is to protect the cerebrum from external insults while regulating the passage of certain substances (e.g., oxygen and nutrients), thereby maintaining normal cerebral function. However, in clinical treatment, the presence of the BBB significantly limits the delivery of therapeutic drugs to the cerebrum. Relevant clinical studies have shown that the rate at which intracerebral neurotransmitters cross the BBB and reach the cerebrum is less than 0.1%. Therefore, the BBB drug delivery barrier has been a long-standing issue in the field of central nervous system (CNS) disease treatment. Summary of the Invention

[0004] The present invention is based, at least in part, on the following discovery: Transporter peptides consisting of the amino acid sequence set forth in SEQ ID NO: 1 have binding affinity for transferrin receptors (TfRs). These transporter peptides effectively bind to transferrin receptors present on tissue barriers and induce cellular transcytosis, allowing these transporter peptides to cross tissue barriers. These transporter peptides can also effectively bind to transferrin receptors on target cells. In some specific embodiments, transporter peptides herein include, but are not limited to, any of the amino acid sequences set forth in SEQ ID NO: 2 to SEQ ID NO: 30, and variants thereof that share at least 70% identity with any of the amino acid sequences set forth in SEQ ID NO: 2 to SEQ ID NO: 30 and are capable of binding to the transferrin receptor.

[0005] In some specific embodiments, the transporter peptides described herein are further conjugated to an effector, either covalently or non-covalently, to form a transporter peptide conjugate. In some specific embodiments, the transporter peptides described herein and the effector form a recombinant transporter peptide conjugate by expressing a peptide encoding the transporter peptide and the effector nucleic acid. These transporter peptides transport the effector to a target by binding to the transferrin receptor. Thus, the transporter peptides described herein can be used as a drug delivery system. Binding of these transporter peptides to the transferrin receptor on cells at a tissue barrier induces the transcytosheath of the cells, thereby transporting the effector conjugated to the transporter peptide and passing through the tissue barrier. The transporter peptides and delivery methods of the present invention effectively cross tissue barriers, particularly the blood-brain barrier, to deliver the effector into the brain. Thus, the transporter peptides and delivery methods of the present invention have broad clinical application value in the prevention and / or treatment of central nervous system diseases.

[0006] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following specific examples.

[0007] Specific Example 1. A transporter peptide consisting of 10 amino acids and having the following general sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 During the ceremony, the first amino acid position (X1) is an acidic amino acid or a polar uncharged amino acid; the second amino acid position (X2) is a nonpolar amino acid; the third amino acid position (X3) is any amino acid; the fourth amino acid position (X4) is an acidic amino acid or a nonpolar amino acid; the fifth amino acid position (X5) is a nonpolar amino acid; the sixth amino acid position (X6) is any amino acid; the seventh amino acid position (X7) is a basic amino acid; the eighth amino acid position (X8) is an acidic amino acid or a basic amino acid; the ninth amino acid position (X9) is a nonpolar amino acid or a polar uncharged amino acid; and The 10th amino acid position (X 10 ) is an acidic amino acid or a polar uncharged amino acid.

[0008] Specific Example 2 A transporter peptide consisting of 10 amino acids and having the following general sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 During the ceremony, the first amino acid position (X1) is Asp, Glu, Asn, Gln, Cys, Ser, Thr, or Tyr; the second amino acid position (X2) is Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp, or Met; the third amino acid position (X3) is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, His, Asp, Asn, Glu, Gln, Lys, Arg, Ser, Thr, Met, Cys, or Pro; the fourth amino acid position (X4) is Asp, Glu, Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp, or Met; the fifth amino acid position (X5) is Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp, or Met; the sixth amino acid position (X6) is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, His, Asp, Asn, Glu, Gln, Lys, Arg, Ser, Thr, Met, Cys, or Pro; the seventh amino acid position (X7) is Lys, His, or Arg; the eighth amino acid position (X8) is Asp, Glu, Lys, His, or Arg; The ninth amino acid position (X9) is Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp, Met, Asn, Gln, Cys, Ser, Thr, or Tyr; and The 10th amino acid position (X 10 ) is Asp, Glu, Asn, Gln, Cys, Ser, Thr or Tyr.

[0009] Specific Example 3. A transporter peptide consisting of 10 amino acids and having the sequence shown in SEQ ID NO: 1 below. X1-Ile-X3-Val-Leu-X6-Lys-X8-X9-X 10 (SEQ ID NO: 1) During the ceremony, the first amino acid position (X1) is Asp, Glu, Asn, Gln, Cys, Ser, Thr, or Tyr; Ile at the second amino acid position (X2) may be replaced by Gly, Ala, Leu, Val, Pro, Phe, Trp, or Met; the third amino acid position (X3) is any amino acid; Val at the fourth amino acid position (X4) may be replaced by Asp, Glu, Gly, Ala, Ile, Leu, Pro, Phe, Trp, or Met; Leu at the fifth amino acid position (X5) may be replaced with Gly, Ala, Ile, Val, Pro, Phe, Trp, or Met; the sixth amino acid position (X6) is any amino acid; Lys at the seventh amino acid position (X7) may be replaced with His or Arg; the eighth amino acid position (X8) is His, Arg, Lys, Asp, or Glu; The ninth amino acid position (X9) is Gly, Ala, Ile, Leu, Val, Pro, Phe, Trp, Met, Asn, Gln, Cys, Ser, Thr, or Tyr; and The 10th amino acid position (X 10 ) is Asp, Glu, Asn, Gln, Cys, Ser, Thr or Tyr.

[0010] Specific Example 4. A transporter peptide according to any one of specific Examples 1 to 3, which is capable of binding to a transferrin receptor (TfR).

[0011] Specific Example 5. The transporter peptide according to any one of Specific Examples 1 to 4, wherein the amino acid sequence is selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30 and mutant sequences that have at least 70% homology to any of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30 and are capable of binding to the transferrin receptor.

[0012] Specific Example 6. The transporter peptide according to specific example 5, wherein the mutant sequence has at least 70% homology with any of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30.

[0013] Specific Example 7. The transporter peptide described in specific example 5, wherein the mutant sequence has at least 80% homology with any of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30.

[0014] Specific Example 8. The transporter peptide according to specific example 5, wherein the mutant sequence has at least 90% homology with any of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30.

[0015] Specific Example 9. A nucleic acid encoding a transporter peptide described in any of Examples 1 to 8.

[0016] Specific Example 10. The nucleic acid of specific example 9, which is deoxyribonucleic acid (DNA).

[0017] Specific Example 11. The nucleic acid of specific example 9, which is ribonucleic acid (RNA).

[0018] Specific Example 12. A vector comprising the nucleic acid described in any of Examples 9 to 11.

[0019] Specific Example 13. The vector described in specific Example 12, which is an adeno-associated viral vector.

[0020] Specific Example 14. A vector as described in specific Example 12, further comprising a nucleic acid encoding an effector.

[0021] Specific Example 15. The vector of specific Example 14, wherein the effector is at least one selected from the group consisting of a peptide, a protein, an antibody, a viral particle, a liposome, an endosome, an exosome, a ligand, a eukaryotic cell, a prokaryotic cell, and a microsphere.

[0022] Specific Example 16. A recombinant transporter peptide conjugate expressed by a vector as described in specific Example 14 or 15.

[0023] Specific Example 17. A recombinant host cell comprising components selected from the group consisting of a peptide described in any of Specific Examples 1 to 8, a nucleic acid described in any of Specific Examples 9 to 11, and a vector described in any of Specific Examples 12 to 15.

[0024] Specific Example 18. A transporter peptide conjugate comprising the transporter peptide described in any one of Examples 1 to 8 and an effector, wherein the transporter peptide and the effector are conjugated in a covalent or non-covalent manner.

[0025] Specific Example 19. 19. The transporter peptide conjugate of specific Example 18, wherein the effector is at least one selected from the group consisting of siRNA, shRNA, microRNA, double-stranded RNA, single-stranded RNA, DNA, oligonucleotide, aptamer, gene, peptide, protein, antibody, small chemical molecule, large chemical molecule, viral particle, liposome, endosome, exosome, nanoparticle, lipid nanoparticle, dendrimer, ligand, eukaryotic cell, prokaryotic cell, microsphere, nanogel, and bio-nanocapsule.

[0026] Specific Example 20. Specific transporter peptides described in any of Examples 1 to 8; A recombinant transporter peptide conjugate as described in specific Example 16; or Transporter peptide conjugates as described in specific Examples 18 or 19 A composition comprising:

[0027] Specific Example 21. The composition of specific example 20 further comprising a pharmaceutically acceptable carrier.

[0028] Specific Example 22. A method for transporting a composition described in specific Example 20 or 21 to a target, comprising binding the transporter peptide, the transporter peptide on the recombinant transporter peptide conjugate, or the transporter peptide on the transporter peptide conjugate to a transferrin receptor.

[0029] Specific Example 23. The method of specific Example 22, wherein the composition must cross a tissue barrier to reach the target and the transferrin receptor is on cells of the tissue barrier.

[0030] Specific Example 24. The method of specific Example 22 or 23, wherein binding of the transporter peptide of the composition, the transporter peptide on a transporter peptide conjugate of the composition, or the transporter peptide on a recombinant transporter peptide conjugate of the composition to the transferrin receptor induces transcytosis of the cell, thereby transporting the composition across the tissue barrier to reach the target.

[0031] Specific Example 25. The method according to any one of Specific Examples 22 to 24, wherein the tissue barriers include the blood-brain barrier (BBB), the mucosal barrier, and the gastrointestinal barrier.

[0032] Specific Example 26. The method according to any of specific Examples 22 to 25, wherein the target is a brain cell.

[0033] Specific Example 27. The method according to any one of specific Examples 22 to 26, wherein the tissue barrier is the blood-brain barrier (BBB) ​​and the target is a brain cell.

[0034] Specific Example 28. The method of specific Example 22, wherein the target is a cell that expresses the transferrin receptor and the transferrin receptor is on the target.

[0035] Specific Example 29. The method according to specific example 22 or 28, wherein the target is a cancer cell.

[0036] Specific Example 30. 29. The method according to specific example 29, wherein the cancer is hepatocellular carcinoma, breast cancer, lung cancer, colon cancer, brain cancer, glioma, prostate cancer, ovarian cancer, or leukemia.

[0037] Specific Example 31. The method according to specific example 22 or 29, wherein the target is a tissue cell.

[0038] Specific Example 32. The method of specific Example 31, wherein the tissue is skin, tonsil, tongue, esophagus, cervix, kidney, placenta, pancreas, testis, anterior pituitary, stomach, breast, or liver.

[0039] Specific Example 33. The method according to any of specific Examples 22-32, wherein the target is inside or outside the body.

[0040] Specific Example 34. A method for transporting an effector across a tissue barrier in a subject, comprising administering to said subject a recombinant transporter peptide conjugate described in specific Example 16, or a transporter peptide conjugate described in specific Example 18 or 19.

[0041] Specific Example 35. The method of specific Example 34, wherein the recombinant transporter peptide conjugate or the transporter peptide conjugate comprises the effector.

[0042] Specific Example 36. The method according to specific example 34 or 35, wherein the tissue barriers include the blood-brain barrier (BBB), the mucosal barrier, and the gastrointestinal barrier.

[0043] Specific Example 37. The method according to any of specific Examples 34-36, wherein the cells of the tissue barrier have transferrin receptors on them.

[0044] Specific Example 38. The method of any of specific Examples 34-37, wherein the transporter peptide binds to the transferrin receptor, and the binding of the transporter peptide to the transferrin receptor induces transcytosheath of the cell, thereby transporting the transporter peptide and the effector across the tissue barrier.

[0045] Specific Example 39. The method according to any of specific Examples 34-38, wherein the administration method includes intradermal, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, intraperitoneal, parenteral, oral, rectal, nasal, pulmonary, and transdermal administration.

[0046] Specific Example 40. The method according to any one of specific Examples 34 to 39, wherein the effector contained in the recombinant transporter peptide conjugate is at least one selected from the group consisting of a peptide, a protein, an antibody, a virus particle, a liposome, an endosome, an exosome, a ligand, a eukaryotic cell, a prokaryotic cell, and a microsphere.

[0047] Specific Example 41. The method according to any one of specific Examples 34 to 39, wherein the effector contained in the recombinant transporter peptide conjugate is at least one selected from the group consisting of siRNA, shRNA, microRNA, double-stranded RNA, single-stranded RNA, DNA, oligonucleotide, aptamer, gene, peptide, protein, antibody, small chemical molecule, large chemical molecule, virus particle, liposome, endosome, exosome, nanoparticle, lipid nanoparticle, dendrimer, ligand, eukaryotic cell, prokaryotic cell, microsphere, nanogel, and bio-nanocapsule.

[0048] Specific Example 42. The method according to any one of specific Examples 34 to 41, wherein the subject is a mammal.

[0049] Specific Example 43. The method of any of specific Examples 34-42, wherein the subject is a rodent or a human subject.

[0050] Specific Example 44. A method for treating and / or preventing a central nervous system (CNS) disease, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant transporter peptide conjugate as described in specific Example 16, or a transporter peptide conjugate as described in specific Example 18 or 19.

[0051] Specific Example 45. The method of specific Example 44, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0052] Specific Example 46. The method according to specific Example 44 or 45, wherein the recombinant transporter peptide conjugate or the effector contained in the transporter peptide conjugate is a therapeutic agent for a central nervous system disease.

[0053] Specific Example 47. The method of any of specific Examples 44 to 46, wherein the transporter peptide on the recombinant transporter peptide conjugate or the transporter peptide on the transporter peptide conjugate binds to a transferrin receptor on cells of a tissue barrier in the body of the test subject, and the binding of the transporter peptide to the transferrin receptor induces transcytosheath of the cells, thereby transporting the transporter peptide and the therapeutic agent for the central nervous system disease through the tissue barrier.

[0054] Specific Example 48. The method according to any of specific Examples 44 to 47, wherein the tissue barriers include the blood-brain barrier, the mucosal barrier, and the gastrointestinal barrier.

[0055] Specific Example 49. The method according to any one of specific Examples 44 to 48, wherein the subject is a mammal.

[0056] Specific Example 50. The method of any of specific Examples 44-49, wherein the subject is a rodent or a human subject.

[0057] Specific Example 51. The method according to any of specific Examples 44 to 50, wherein the administration method includes intradermal, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, intraperitoneal, parenteral, oral, rectal, nasal, pulmonary, and transdermal administration.

[0058] Specific Example 52. The method according to any one of specific Examples 44 to 51, wherein the central nervous system disease comprises Alzheimer's disease (AD), Parkinson's disease (PD), cerebrovascular accidents (CVA), vascular-related dementia, Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), traumatic brain injury (TBI), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington's disease, and spinal muscular atrophy (SMA).

[0059] Specific Example 53. Specific examples of the use of a transporter peptide according to any one of Examples 1 to 8 in transporting an effector to a target.

[0060] Specific Example 54. Specific examples of the use of a transporter peptide according to any one of Examples 1 to 8 in the preparation of a pharmaceutical composition for transporting an effector to a target.

[0061] Specific Example 55. The use of specific example 53 or 54, wherein the transporter peptide and the effector are conjugated in a covalent or non-covalent manner to form a transporter peptide conjugate of specific example 18 or 19.

[0062] Specific Example 56. The use of specific example 53 or 54, wherein the transporter peptide and the effector form a recombinant transporter peptide conjugate as described in specific example 16, such that nucleic acids encoding the transporter peptide and the effector are expressed.

[0063] Specific Example 57. The use according to any of specific Examples 53 to 56, wherein the transporter peptide and the effector must cross a tissue barrier to reach the target, and the transporter peptide binds to a transferrin receptor on cells of the tissue barrier.

[0064] Specific Example 58. The use described in specific Example 57, wherein binding of the transporter peptide to the transferrin receptor induces transcytosheathment of the cell, thereby transporting the transporter peptide and the effector across the tissue barrier to reach the target.

[0065] Specific Example 59. The use according to specific example 57 or 58, wherein the tissue barriers include the blood-brain barrier, the mucosal barrier, and the gastrointestinal barrier.

[0066] Specific Example 60 The use described in any of Examples 53 to 56, wherein the target is a cell expressing a transferrin receptor, and the transporter peptide transports the effector to the target by binding to the transferrin receptor expressed on the target cell.

[0067] Specific Example 61. The use according to specific example 60, wherein the target is a cancer cell or tissue cell.

[0068] Specific Example 62. The use according to specific Example 61, wherein the cancer is liver cancer, breast cancer, lung cancer, colon cancer, brain cancer, glioma, prostate cancer, ovarian cancer, or leukemia.

[0069] Specific Example 63. The use according to specific Example 61, wherein the tissue is skin, tonsils, tongue, esophagus, cervix, kidney, placenta, pancreas, testis, anterior pituitary gland, stomach, breast, or liver.

[0070] Specific Example 64. The use according to any of specific Examples 53 to 63, wherein the target is inside or outside the body.

[0071] Specific Example 65. Specific examples of use of a transporter peptide as described in any one of Examples 1 to 8 in transporting an effector across a tissue barrier in a subject.

[0072] Specific Example 66. Specific examples of use of a transporter peptide as described in any one of Examples 1 to 8 in the preparation of a pharmaceutical composition for transporting an effector across a tissue barrier in a subject.

[0073] Specific Example 67. The use of specific example 65 or 66, wherein the transporter peptide and the effector are conjugated in a covalent or non-covalent manner to form a transporter peptide conjugate of specific example 18 or 19.

[0074] Specific Example 68. The use of specific example 65 or 66, wherein the transporter peptide and the effector form a recombinant transporter peptide conjugate as described in specific example 16, such that nucleic acids encoding the transporter peptide and the effector are expressed.

[0075] Specific Example 69. The use according to any of specific Examples 65 to 68, wherein the tissue barriers include the blood-brain barrier, the mucosal barrier, and the gastrointestinal barrier.

[0076] Specific Example 70. The use according to any of specific Examples 65 to 69, wherein the cells of the tissue barrier have transferrin receptors on them.

[0077] Specific Example 71. The use of any of specific Examples 65-70, wherein the transporter peptide binds to the transferrin receptor, and the binding of the transporter peptide to the transferrin receptor induces transcytosheathment of the cell, thereby transporting the transporter peptide and the effector across the tissue barrier.

[0078] Specific Example 72. Specific examples of the use of a transporter peptide according to any one of Examples 1 to 8 in the treatment and / or prevention of central nervous system disorders.

[0079] Specific Example 73. Use of a transporter peptide according to any of specific Examples 1 to 8 in the preparation of a pharmaceutical composition for treating and / or preventing central nervous system diseases.

[0080] Specific Example 74. The use of specific example 72 or 73, wherein the transporter peptide and the effector are conjugated, either covalently or non-covalently, to form the transporter peptide conjugate of specific example 18 or 19.

[0081] Specific Example 75. The use of specific example 72 or 73, wherein the transporter peptide and the effector form a recombinant transporter peptide conjugate as described in specific example 16, such that nucleic acids encoding the transporter peptide and the effector are expressed.

[0082] Specific Example 76. The use according to specific example 74 or 75, wherein said effector is a therapeutic agent for treating a central nervous system disorder.

[0083] Specific Example 77. The use according to any of specific Examples 74 to 76, wherein the transporter peptide conjugate or the recombinant transporter peptide conjugate is administered to a subject in need thereof.

[0084] Specific Example 78. The use described in specific Example 77, wherein the transporter peptide binds to a transferrin receptor on cells of a tissue barrier within the subject's body, and the binding of the transporter peptide to the transferrin receptor induces transcytosheathing of the cells, thereby transporting the transporter peptide and the therapeutic agent for the central nervous system disorder across the tissue barrier.

[0085] Specific Example 79. The use according to specific Example 78, wherein the tissue barriers include the blood-brain barrier, the mucosal barrier, and the gastrointestinal barrier.

[0086] Specific Example 80. The use according to any of specific Examples 72 to 79, wherein the central nervous system disease comprises Alzheimer's disease, Parkinson's disease, cerebrovascular disease, vascular dementia, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, and spinal muscular atrophy.

[0087] Specific Example 81. The use according to any one of specific Examples 53 to 55, 57 to 67, 69 to 74, and 76 to 80, wherein the effector is at least one selected from the group consisting of siRNA, shRNA, microRNA, double-stranded RNA, single-stranded RNA, DNA, oligonucleotide, aptamer, gene, peptide, protein, antibody, small chemical molecule, large chemical molecule, virus particle, liposome, endosome, exosome, nanoparticle, lipid nanoparticle, dendrimer, ligand, eukaryotic cell, prokaryotic cell, microsphere, nanogel, and bio-nanocapsule.

[0088] Specific Example 82. The use according to any one of specific Examples 53 to 54, 56 to 66, 68 to 73, and 75 to 80, wherein the effector is at least one selected from the group consisting of a peptide, a protein, an antibody, a virus particle, a liposome, an endosome, an exosome, a ligand, a eukaryotic cell, a prokaryotic cell, and a microsphere.

[0089] Specific Example 83. The use according to any of specific Examples 65 to 82, wherein the subject is a mammal.

[0090] Specific Example 84. The use according to any of specific Examples 65 to 83, wherein the subject is a rodent or a human subject.

[0091] These and other aspects will become apparent from the following description of the preferred specific embodiments, taken in conjunction with the drawings.

[0092] The drawings illustrate one or more illustrative embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Where possible, the same drawing reference numerals will be used throughout the drawings to refer to the same or similar parts in the illustrative embodiments. [Brief explanation of the drawings]

[0093] [Figure 1]1 shows experimental results regarding the transport efficiency of a transporter peptide (PT-034, SEQ ID NO: 19) for Caco-2 monolayer cells. [Figure 2A] FIG. 1 shows in vivo fluorescence imaging images of the transporter peptide of the present invention (PT-034, SEQ ID NO: 19) administered to mice 1, 2, 4, 6, and 24 hours after administration. [Figure 2B] Quantitative fluorescence intensity plots for mouse brain tissue. [Figure 3A] In vivo fluorescence imaging images taken 0.5, 1, 2, 4, 6, and 24 hours after administration of the transporter peptides of the present invention (PT-001, SEQ ID NO: 2, PT-025, SEQ ID NO: 11, or PT-031, SEQ ID NO: 17) to mice. [Figure 3B] Quantitative fluorescence intensity plot for mouse brain tissue. [Figure 4A] 1 shows in vivo fluorescence imaging images taken 0.5, 1, 2, 4, 6, and 24 hours after administration of the transporter peptide of the present invention (PT-034 and antibody conjugate) to mice. [Figure 4B] Quantitative fluorescence intensity plot for mouse brain tissue. [Figure 5A] Figure 1 shows in vivo fluorescence imaging images taken 0.5, 1, 2, 4, 6, and 24 hours after intravenous injection or oral administration of the transporter peptide conjugate of the present invention (a conjugate of PT-034 and an antibody) to mice. [Figure 5B] Quantitative fluorescence intensity plot for mouse brain. [Figure 5C] Quantitative fluorescence intensity plot for mouse brain tissue. [Figure 6A] In vivo fluorescence imaging images taken 0.5, 1, 2, 4, 6, and 24 hours after administration of the transporter peptide conjugate of the present invention (a conjugate of PT-034 and lipid nanoparticles) to mice. [Figure 6B] Quantitative fluorescence intensity plot for mouse brain tissue. DETAILED DESCRIPTION OF THE INVENTION

[0094] The present invention is based, at least in part, on the following discovery: The transporter peptides of the present invention can effectively bind to transferrin receptors on cells. In some specific embodiments, the transporter peptides of the present invention can be conjugated to a substance, either covalently or non-covalently, to form a transporter peptide conjugate. In some specific embodiments, the transporter peptides of the present invention and an effector form a recombinant transporter peptide conjugate, such that the cell expresses nucleic acids encoding the transporter peptide and the effector. When the cell is a cell on a tissue barrier, binding of the transporter peptide of the present invention to the transferrin receptor induces a transcytosheath in the cell, thereby allowing a substance conjugated to the transporter peptide of the present invention to pass through the tissue barrier. When the cell is a target cell, binding of the transporter peptide of the present invention to the transferrin receptor can transport the conjugated substance to the target cell. Thus, the transporter peptides of the present invention can be used as a drug delivery system. In particular, the transporter peptides of the present invention can deliver a substance conjugated to the transporter peptide of the present invention into the brain by transporting it through the transcytosheath and passing through a tissue barrier, particularly the blood-brain barrier. Therefore, the transporter peptide according to the present invention can be used for the prevention and / or treatment of central nervous system diseases.

[0095] Specifically, the present invention provides transporter peptides consisting of 10 amino acids and having the following general formula: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 During the ceremony, the first amino acid position (X1) is an acidic amino acid (Asp or Glu) or a polar uncharged amino acid (Asn, Gln, Cys, Ser, Thr, or Tyr); the second amino acid position (X2) is a nonpolar amino acid (Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp, or Met); the third amino acid position (X3) is any amino acid (Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, His, Asp, Asn, Glu, Gln, Lys, Arg, Ser, Thr, Met, Cys, or Pro); the fourth amino acid position (X4) is an acidic amino acid (Asp or Glu) or a nonpolar amino acid (Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp, or Met); the fifth amino acid position (X5) is a nonpolar amino acid (Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp, or Met); the sixth amino acid position (X6) is any amino acid (Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, His, Asp, Asn, Glu, Gln, Lys, Arg, Ser, Thr, Met, Cys, or Pro); the seventh amino acid position (X7) is a basic amino acid (Lys, His, or Arg); the eighth amino acid position (X8) is an acidic amino acid (Asp or Glu) or a basic amino acid (Lys, His, or Arg); The ninth amino acid position (X9) is a nonpolar amino acid (Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp, or Met) or a polar uncharged amino acid (Asn, Gln, Cys, Ser, Thr, or Tyr); and The 10th amino acid position (X 10 ) is an acidic amino acid (Asp or Glu) or a polar uncharged amino acid (Asn, Gln, Cys, Ser, Thr, or Tyr).

[0096] More specifically, the transporter peptide provided by the present invention preferably has the sequence shown in SEQ ID NO: 1 below. X1-Ile-X3-Val-Leu-X6-Lys-X8-X9-X 10 (SEQ ID NO: 1) During the ceremony, the first amino acid position (X1) is Asp, Glu, Asn, Gln, Cys, Ser, Thr, or Tyr; Ile at the second amino acid position (X2) may be replaced by Gly, Ala, Leu, Val, Pro, Phe, Trp, or Met; the third amino acid position (X3) is any amino acid; Val at the fourth amino acid position (X4) may be replaced by Asp, Glu, Gly, Ala, Ile, Leu, Pro, Phe, Trp, or Met; Leu at the fifth amino acid position (X5) may be replaced with Gly, Ala, Ile, Val, Pro, Phe, Trp, or Met; the sixth amino acid position (X6) is any amino acid; Lys at the seventh amino acid position (X7) may be replaced with His or Arg; the eighth amino acid position (X8) is His, Arg, Lys, Asp, or Glu; The ninth amino acid position (X9) is Gly, Ala, Ile, Leu, Val, Pro, Phe, Trp, Met, Asn, Gln, Cys, Ser, Thr, or Tyr; and The 10th amino acid position (X 10 ) is Asp, Glu, Asn, Gln, Cys, Ser, Thr or Tyr.

[0097] In some preferred specific embodiments, the amino acid sequence of SEQ ID NO: 1 includes, but is not limited to, the amino acid sequences of SEQ ID NO: 2 through SEQ ID NO: 30, and variants thereof that share at least 70% identity with any of the amino acid sequences of SEQ ID NO: 2 through SEQ ID NO: 30 and are capable of binding to the transferrin receptor. In some preferred specific embodiments, the variants share at least 70% identity with any of the amino acid sequences of SEQ ID NO: 2 through SEQ ID NO: 30. In some preferred specific embodiments, the variants share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with any of the amino acid sequences of SEQ ID NO: 2 through SEQ ID NO: 30. Each possibility represents a separate embodiment of the present invention. The sequences of SEQ ID NO: 2 to SEQ ID NO: 30 are shown in Table 1 below.

[0098] To determine the percentage of homology between two sequences, the sequences are compared to achieve the goal of best matching (e.g., introducing blanks into the first amino acid sequence to achieve the best match with the second amino acid sequence). When calculating the percentage of homology, an exact match is usually calculated. The percentage of homology or identity between two sequences may also be determined using mathematical algorithms well known in the art, such as BLAST and Gapped BLAST programs, NBLAST and XBLAST programs, or the ALIGN program.

[0099] TIFF2025538660000002.tif222151

[0100] In some preferred specific embodiments, the amino acid sequence shown in SEQ ID NO: 1 has binding affinity to the transferrin receptor.

[0101] The term "peptide" as used herein refers to a molecular chain of amino acids, including L- and D-forms. If necessary, amino acids can be modified in vivo or in vitro, for example, by mannosylation, glycosylation, amidation (especially C-terminal amide), carboxylation, or phosphorylation, provided that these modifications maintain the biological activity of the original molecule. Peptides may also be part of fusion proteins. The term "transporter peptide" as used herein describes the application of peptides and refers to peptides that can induce transport effects by binding to a specific receptor, and is also a target peptide.

[0102] Functional derivatives of peptides are also encompassed by the present invention. Functional derivatives encompass peptides with one or more amino acid deletions, substitutions, inversions, or additions throughout the sequence. Amino acid substitutions that are not expected to fundamentally alter biological and immunological activity have been described. Amino acid substitutions between related amino acids, or substitutions that have commonly occurred in evolution, include Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, and Ile / Val.

[0103] The peptides of the present invention can be produced synthetically or by recombinant DNA techniques. Methods for producing synthetic peptides are well known in the art.

[0104] The nomenclature used herein to describe the peptides of the invention follows normal practice, with the amino group (N-terminus) and / or 5'-terminus on the left and the carboxyl group (C-terminus) and / or 3'-terminus on the right. As used herein, the term "amino acid position" refers to the amino acid at a given position in the peptide, calculated from the N-terminus of the peptide; for example, the first amino acid position (X1) refers to the first amino acid position counting from the N-terminus of the peptide.

[0105] The term "any amino acid" as used herein refers to any one amino acid selected from the 20 basic amino acids. The term "basic amino acid" as used herein includes glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, Ile), phenylalanine (Phe, F), tyrosine (Tyrosine, Tyr, Y), tryptophan (Trp, W), histidine (Histidine, His, H), aspartic acid (Asp, D), asparagine (Asparagine, Asn, N), and glutamic acid (Glutamic acid). The amino acids include 20 amino acids: Glu (Glu, E), Glutamine (Gln, Q), Lysine (Lys, K), Arginine (Arg, R), Serine (Ser, S), Threonine (Thr, T), Methionine (Methionine, Met, M), Cysteine ​​(Cys, C), and Proline (Pro, P). The symbols used herein to represent amino acids are the same as the abbreviations used by those skilled in the art. Unless otherwise defined, as used herein, the term "acidic amino acid" refers to Asp or Glu, the term "basic amino acid" refers to His, Arg, or Lys, the term "polar uncharged amino acid" refers to Asn, Gln, Cys, Ser, Thr, or Tyr, and the term "nonpolar amino acid" refers to Gly, Ala, Ile, Leu, Val, Pro, Phe, Trp, or Met. Unless an amino acid is specifically designated as being dextrorotatory or levorotatory, the amino acid may be a levorotatory amino acid, or a levorotatory or dextrorotatory amino acid, unless the context clearly indicates that the amino acid is a particular isomer.

[0106] As used herein, the term "transferrin receptor" (TfR) refers to a type II transmembrane glycoprotein with a molecular weight of 90 kDa that is found as a disulfide-bonded homodimer (180 kDa) on the surface of cells in which it resides. The protein sequence of the transferrin receptor is available from the Uniprot website under protein number P02786. The extracellular domain of each transferrin receptor consists of three domains: the top domain (amino acid residues 189-383), the protease-like domain (amino acid residues 122-188 and 384-606), and the coil domain (amino acid residues 607-760). The transferrin-binding domain is primarily located in the coil domain and a portion of the protease-like domain on the surface of the transferrin receptor. The primary role of the transferrin receptor is to bind to transferrin (Fe-transferrin, Tf, a protein that transports iron in the blood) and promote cellular iron absorption. Binding of the transferrin receptor to transferrin induces transcytosis of the cell where the transferrin receptor is located, thereby transporting the transferrin and the iron it carries into the cell. Transferrin receptors are commonly found on various tissue barriers, including the blood-brain barrier, gastrointestinal barrier, and mucosal barrier. Furthermore, transferrin receptors have been found to be relatively highly expressed in rapidly proliferating cells (e.g., cancer cells), such as those associated with liver cancer, breast cancer, lung cancer, colon cancer, brain tumors, gliomas, prostate cancer, ovarian cancer, and leukemia. Transferrin receptors have also been found to be expressed in cells of certain tissues, including, but not limited to, skin, tonsils, tongue, esophagus, cervix, kidney, placenta, pancreas, testis, anterior pituitary gland, stomach, breast, and liver.

[0107] The present invention also provides nucleic acids encoding the transporter peptides described herein. In some preferred embodiments, the nucleic acid is deoxyribonucleic acid (DNA). In some preferred embodiments, the nucleic acid is ribonucleic acid (RNA).

[0108] As used herein, the term "nucleotide" refers to a nitrogenous base attached to a sugar phosphate, including sugars such as ribose or 2'-deoxyribose, with one or more phosphate groups attached to the sugar. "Polynucleotide" and "nucleic acid" refer to polymers of more than one nucleotide unit, where the units are typically joined by sugar-phosphate bonds in a sugar-phosphate backbone. A polynucleotide does not necessarily contain a single type of nucleotide unit. For example, a given polynucleotide may contain only ribonucleotides, only 2'-deoxyribonucleotides, or a combination of ribonucleotides and 2'-deoxyribonucleotides. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as nucleic acid analogs containing one or more non-natural units. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this will be understood to also include RNA sequences in which "U" replaces "T" (i.e., A, U, G, C). The term "cDNA" refers to DNA complementary to or identical to mRNA, in which "T" replaces "U", and may be in single- or double-stranded form. The term "recombinant nucleic acid" refers to a polynucleotide or nucleic acid having a non-naturally linked sequence. Recombinant nucleic acids may be in the form of a vector.

[0109] The nucleic acids encoding any transporter peptide herein are derived from the amino acid sequence of the transporter peptide of the present invention. The nucleic acid sequences provided by the present invention can be obtained by substituting each amino acid in the amino acid sequence of the transporter peptide of the present invention with a nucleotide sequence (including degenerate codons, also known as synonymous codons) encoding that amino acid listed in the genetic code table. For example, proline in the amino acid sequence of the transporter peptide of the present invention is encoded by a nucleotide sequence such as CCA, CCC, CCG, or CCT.

[0110] The present invention also provides a vector comprising a nucleic acid encoding the transporter peptide described herein. In some preferred specific embodiments, the vector is an adeno-associated virus vector. In some preferred specific embodiments, the vector further comprises a nucleic acid encoding an effector. In some more preferred specific embodiments, the effector is at least one selected from the group consisting of a peptide, a protein, an antibody, a virus particle, a liposome, an endosome, an exosome, a ligand, a eukaryotic cell, a prokaryotic cell, and a microsphere.

[0111] As used herein, the term "vector" refers to a DNA molecule that incorporates exogenous DNA into a host organism (usually a bacterial, yeast, or mammalian cell). Examples of vectors include, but are not limited to, plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), sticky particles, shuttle vectors, expression vectors, retroviral vectors, and adenoviral vectors.

[0112] As used herein, the term "adeno-associated virus (AAV)" refers to a small, non-enveloped virus belonging to the Parvoviridae family and Dependoparvovirus genus. Because AAV does not replicate in infected cells, it is not associated with any known human diseases and is considered relatively safe for use as a gene delivery vector. As used herein, the term "adeno-associated virus vector" refers to a vector obtained by removing most of the viral genes in wild-type adeno-associated virus and replacing these genes with target genes. AAV vectors are used to introduce a desired gene or genetic material into target cells or tissues and are commonly used in gene therapy and molecular biology research.

[0113] As used herein, the term "effector" refers to any molecule that has a function on a target within a tissue barrier, which may be a target cell or an extracellular molecule.

[0114] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides containing at least one binding domain, formed by folding of the polypeptide chain into a three-dimensional binding cavity with an internal shape and charge distribution complementary to the epitope characteristics of an antigen. Antibodies typically have a tetrameric format, comprising two pairs of identical polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form an antibody binding site. Antibodies may be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, polyspecific, bispecific, catalytic, humanized, fully humanized, or anti-genotype antibodies and fragments that can be marked in soluble or binding form, including epitope-binding fragments, variants, or derivatives, either alone or in combination with other amino acid sequences. Antibodies can be derived from any species. The term antibody also encompasses binding fragments, including, but not limited to, Fv, Fab, Fab', F(ab')2, single-stranded antibodies (svFC), dimeric variable regions (diabodies), and disulphide-linked variable regions (dsFv). Specifically, antibodies encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain one antigen-binding site. Antibody fragments may or may not be fused to domains of other immunoglobulins, and include, but are not limited to, Fc regions or fragments thereof. Those skilled in the art will appreciate that other fusion products can be generated, such as scFv-Fc fusions, variable regions (e.g., V L and V H It will be further understood that the term "antibody" includes, but is not limited to, scFv-scFv-Fc fusions and scFv-scFv-Fc fusions.

[0115] The present invention also provides recombinant transporter peptide conjugates obtained by expression of the vectors herein.

[0116] The present invention also provides a recombinant host cell comprising a component selected from any one of the peptides herein, the nucleic acids herein, and the vectors herein.

[0117] The present invention also provides a transporter peptide conjugate comprising any one of the transporter peptides described herein and an effector covalently or non-covalently conjugated to the transporter peptide. In some preferred embodiments, the effector is at least one selected from the group consisting of siRNA, shRNA, microRNA, double-stranded RNA, single-stranded RNA, DNA, oligonucleotide, aptamer, gene, peptide, protein, antibody, small chemical molecule, large chemical molecule, virus particle, liposome, endosome, exosome, nanoparticle, lipid nanoparticle, dendrimer, ligand, eukaryotic cell, prokaryotic cell, microsphere, nanogel, and bio-nanocapsule.

[0118] The present invention also provides compositions comprising at least one of the transporter peptides described herein, the recombinant transporter peptide conjugates described herein, and the transporter peptide conjugates described herein, which, in some preferred embodiments, further comprise a pharmaceutically acceptable carrier.

[0119] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and other physiologically acceptable excipients or additives. In certain specific embodiments, the carrier is suitable for nasal, intravenous, intramuscular, intradermal, subcutaneous, parenteral, oral, transmucosal, and transdermal administration. Depending on the route of administration, the active compound may be enclosed in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The application of such media and agents to pharmaceutical active substances is well known in the art.

[0120] The present invention also provides a method for transporting a composition described herein to a target, comprising binding the transporter peptide, the transporter peptide on the recombinant transporter peptide conjugate, or the transporter peptide on the transporter peptide conjugate to a transferrin receptor. The present invention also provides a use of the transporter peptide described herein in transporting an effector to a target. The present invention also provides a use of the transporter peptide described herein in producing a pharmaceutical composition that transports an effector to a target, wherein the target is inside or outside the body.

[0121] In some preferred specific embodiments, the composition must cross a tissue barrier to reach the target, and the transferrin receptor is present on cells of the tissue barrier. In some preferred specific embodiments, binding of the transporter peptide of the composition, the transporter peptide on a transporter peptide conjugate of the composition, or the transporter peptide on a recombinant transporter peptide conjugate of the composition to the transferrin receptor induces transcytosis of the cells, thereby transporting the composition across the tissue barrier and reaching the target. In some preferred specific embodiments, the tissue barrier includes the blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier. In some preferred specific embodiments, the target is a brain cell. In some more preferred specific embodiments, the tissue barrier is the blood-brain barrier and the target is a brain cell.

[0122] In some preferred specific embodiments, the target is a cell that expresses the transferrin receptor, and the transferrin receptor is on the target. In some preferred specific embodiments, the target is a cancer cell. In some more preferred specific embodiments, the cancer is liver cancer, breast cancer, lung cancer, colon cancer, brain cancer, glioma, prostate cancer, ovarian cancer, or leukemia. In some preferred specific embodiments, the target is a tissue cell. In some more preferred specific embodiments, the tissue is skin, tonsil, tongue, esophagus, cervix, kidney, placenta, pancreas, testis, anterior pituitary gland, stomach, breast, or liver.

[0123] The present invention also provides a method for transporting an effector across a tissue barrier in a subject, comprising administering to said subject a recombinant transporter peptide conjugate as described herein, or a transporter peptide conjugate as described herein; the use of a transporter peptide as described herein in transporting an effector across a tissue barrier in a subject; and the use of a transporter peptide as described herein in the preparation of a pharmaceutical composition for transporting an effector across a tissue barrier in a subject.

[0124] In some preferred specific embodiments, the recombinant transporter peptide conjugate or the transporter peptide conjugate comprises the effector. In some preferred specific embodiments, the tissue barrier includes the blood-brain barrier, mucosal barrier, and gastrointestinal barrier. In some preferred specific embodiments, transferrin receptors are present on cells of the tissue barrier. In some preferred specific embodiments, the transporter peptide binds to the transferrin receptor, and binding of the transporter peptide to the transferrin receptor induces transcytosheathing of the cells, thereby transporting the transporter peptide and the effector across the tissue barrier. In some preferred specific embodiments, the administration method includes intradermal, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, intraperitoneal, parenteral, oral, rectal, nasal, pulmonary, and transdermal administration. In some preferred specific embodiments, the effector contained in the recombinant transporter peptide conjugate is at least one selected from the group consisting of a peptide, a protein, an antibody, a virus particle, a liposome, an endosome, an exosome, a ligand, a eukaryotic cell, a prokaryotic cell, and a microsphere. In some preferred specific embodiments, the effector contained in the recombinant transporter peptide conjugate is at least one selected from the group consisting of siRNA, shRNA, microRNA, double-stranded RNA, single-stranded RNA, DNA, an oligonucleotide, an aptamer, a gene, a peptide, a protein, an antibody, a small chemical molecule, a large chemical molecule, a virus particle, a liposome, an endosome, an exosome, a nanoparticle, a lipid nanoparticle, a dendrimer, a ligand, a eukaryotic cell, a prokaryotic cell, a microsphere, a nanogel, and a bio-nanocapsule. In some preferred specific embodiments, the subject is a mammal. In some more preferred specific embodiments, the subject is a rodent or a human subject.

[0125] The present invention also provides a method for treating and / or preventing a central nervous system (CNS) disorder, comprising administering a therapeutically effective amount of a recombinant transporter peptide conjugate herein, or a transporter peptide conjugate herein, to a subject in need thereof. The present invention also provides a use of a transporter peptide herein in treating and / or preventing a central nervous system disorder. The present invention also provides a use of a transporter peptide herein in preparing a pharmaceutical composition for treating and / or preventing a central nervous system disorder.

[0126] In some preferred specific embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some preferred specific embodiments, the recombinant transporter peptide conjugate or the effector contained in the transporter peptide conjugate is a therapeutic agent for a central nervous system disease. In some preferred specific embodiments, the transporter peptide on the recombinant transporter peptide conjugate or the transporter peptide on the transporter peptide conjugate binds to a transferrin receptor on cells of a tissue barrier in the subject's body, and the binding of the transporter peptide to the transferrin receptor induces transcytosheathing of the cells, thereby transporting the transporter peptide and the therapeutic agent for a central nervous system disease across the tissue barrier. In some preferred specific embodiments, the tissue barrier includes the blood-brain barrier, mucosal barrier, and gastrointestinal barrier. In some preferred specific embodiments, the subject is a mammal. In some more preferred specific embodiments, the subject is a rodent or a human subject. In some preferred embodiments, the administration method includes intradermal, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, intraperitoneal, parenteral, oral, rectal, nasal, pulmonary, and transdermal administration. In some preferred embodiments, the central nervous system disease includes Alzheimer's disease, Parkinson's disease, cerebrovascular disease, vascular dementia, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, and spinal muscular atrophy.

[0127] Formulations suitable for administration according to the present invention can include aqueous and non-aqueous solutions, antioxidants, bacteriostats, buffers, solutes that affect isotonicity, preservatives, solubilizers, stabilizers, suspending agents, thickening agents, or combinations thereof, among other factors known to those skilled in the art.

[0128] Additionally or alternatively, formulations suitable for administration of the present invention can include gels, PEG (e.g., PEG 400), propylene glycol, saline, packets, water, other suitable liquids known in the art, or combinations thereof, among other circumstances known to those of skill in the art.

[0129] Additionally or alternatively, formulations suitable for administration of the present invention can include adhesives, buffers, tricalcium phosphate, cellulose, colloids (e.g., colloidal silicon dioxide), colorants, diluents, disintegrants, dyes, fillers, flavorings, gelatin, lactose, magnesium stearate, mannitol, microcrystalline gelatin, humectants, paraffin hydrocarbons, tablets, polyethylene glycol, preservatives, sorbitol, starch (e.g., corn starch, potato starch, or combinations thereof), stearic acid, sucrose, talc, triglycerides, or combinations thereof, among other circumstances known to those of skill in the art.

[0130] Additionally or alternatively, formulations suitable for administration of the present invention can include alcohol (e.g., phenylmethanol or ethanol), benzalkonium chloride, a buffer (e.g., phosphate buffer, acetate buffer, citrate buffer, or a combination thereof), carboxymethylcellulose or microcrystalline cellulose, cholesterol, glucose, fruit juice (e.g., yuzu juice, milk), phospholipids (e.g., lecithin), oil (e.g., vegetable oil, fish oil, or mineral oil, or a combination thereof); other pharmaceutically acceptable carriers known in the art; or combinations thereof, among other circumstances known to those of skill in the art.

[0131] Additionally or alternatively, formulations suitable for administration of the present invention can include biodegradable materials (e.g., polylactic-co-glycolic acid (PLGA) polymers), other substantial degradation products that can be rapidly removed from a biological system, or combinations thereof, among other circumstances known to those skilled in the art.

[0132] The formulations of the present invention can be administered in unit dose form, multi-dose form, or a combination thereof. Such formulations can be packaged in unit dose containers, multi-dose containers, or a combination thereof. The present invention can be present in ampoules, cachets, capsules, granules, lozenges, powders, tablets, vials, emulsions (including, but not limited to, gum acacia emulsions), suspensions, or combinations thereof.

[0133] As used herein, the term "effective amount" or "sufficient amount" of a substance refers to an amount sufficient to achieve beneficial or desired results (including clinical results). Thus, an "effective amount" is determined by the context in which it is applied. When administering an immunogenic composition, the effective amount is an immunogenically effective amount, including an amount of the immunogenic composition of the present invention sufficient to elicit an immune response. When administering a drug composition, the effective amount is a pharmaceutically effective amount, including an amount of the drug composition of the present invention sufficient to maintain or produce the desired physiological result. An effective amount can be administered in one or more doses.

[0134] As used herein, the term "pharmaceutically effective amount" refers to an amount capable or sufficient to maintain or produce a desired physiological result, including, but not limited to, treating, reducing, alleviating, eliminating, suppressing, essentially preventing, or preventing a disease, condition, or combination thereof. A pharmaceutically effective amount may include administering one or more doses before, during, or simultaneously. One skilled in the art will understand how to adjust the dosages of the present invention to suit each type of formulation, including, but not limited to, sustained release formulations. As used herein, the term "prophylactic" refers to a composition that can essentially prevent or prevent all aspects of a disease, condition, or combination thereof. As used herein, the term "therapeutic" refers to a composition that can treat, reduce, prevent the progression of, alleviate the progression of, beneficially alter, eliminate, or a combination thereof, all aspects of a disease, condition, or combination thereof.

[0135] As used herein, the term "dose" of a composition refers to a measured portion of that composition taken (administered or received) by a subject at any given time.

[0136] As used herein, the term "subject" refers to animals, and more specifically, to non-human mammals and human organisms. Non-human animal subjects can also include prenatal forms of animals, such as embryos or fetuses. Non-limiting examples of non-human animals include horses, cows, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, and pigs. In some specific embodiments, the subject is a human. Human subjects can also include fetuses.

[0137] As used herein, the term "subject" refers to any subject in need of treatment, particularly mammalian subjects, such as humans.

[0138] As used herein, the terms "treat," "treating," or "treatment" include alleviating at least one symptom, reducing its severity, or inhibiting its worsening. Treating does not necessarily indicate a complete cure of the disease, disorder, or condition. To be an effective treatment, a composition useful herein may reduce the severity of the disease, condition, or condition, reduce the severity of symptoms associated therewith, or improve the quality of life of the patient or subject.

[0139] As used herein, the terms "prevent", "preventing" or "prevention" refer to the ability to essentially eliminate, avoid, evade, forestall, arrest, hinder, impede, or any combination thereof the occurrence of any aspect of a disease, symptom or combination thereof, especially by taking action in advance.

[0140] In some specific embodiments, oligonucleotide therapeutic agents and / or compositions of the present invention can be administered to a subject by multiple routes, including intradermal, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, intraperitoneal, parenteral, oral, rectal, nasal, intrapulmonary, and transdermal administration, or topical administration to the eye, ear, skin, or mucous membranes. Alternatively, the antigen can be optionally carried in a biologically suitable liquid or solid carrier and administered ex vivo by direct contact with cells, tissues, or organs from the subject (autologous) or another subject (alien).

[0141] The meanings of the technical and scientific terms used herein can be clearly understood by those skilled in the art.

[0142] As used herein, the terms "about," "approximately," or "generally," when used in conjunction with a numerical value, refer to plus or minus 10% of the referenced numerical value. For example, a length of about 1000 nanometers (nm) refers to a length in the range of 900 nm to 1100 nm.

[0143] As used herein, the term "comprising" is open, indicating that such embodiment may include additional elements. Conversely, the term "consisting of" is closed, indicating that such embodiment does not include additional elements (except for trace impurities). "Consisting essentially of" is part-closed, indicating that such embodiment may also include elements that do not materially alter the basic characteristics of such embodiment.

[0144] It will be readily understood that when an applicant defines an invention or part thereof using an open conjunction such as "comprising," the specification should be construed as describing the invention also using the conjunctions "consisting essentially of" or "consisting of" (unless otherwise stated).

[0145] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "polynucleotide" includes a plurality of such polynucleotides; a reference to "the polynucleotide" includes one or more of the polynucleotides referenced, as well as equivalents known to those of skill in the art. It should further be noted that the claims may be drafted to exclude all optional elements. Thus, the text is intended to precede the recitation of elements in the claims or, when using a "negative" limitation, the use of exclusive terminology such as "solely," "only," etc.

[0146] In some situations, when a convention similar to "at least one of A, B, and C, etc." is used, such construction is typically intended to ensure that one of ordinary skill in the art understands the meaning of that convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, A and B, A and C, B and C, and / or A, B and C, etc.). Those of ordinary skill in the art will further understand that all disjunctions and / or words that actually represent two or more alternative terms, whether in the description, claims, or drawings, should be considered to encompass the possibilities of one, either, or both of those terms. For example, the words "A or B" are understood to encompass the possibilities of "A," "B," or "A or B."

[0147] The present invention is further illustrated by the following examples. The presentation of these examples is intended to be illustrative and not limiting. Those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific examples disclosed and still obtain like or similar results without departing from the spirit and scope of the present invention. [Example]

[0148] All animal experiment protocols in the examples published in this paper were reviewed and approved by the Institutional Animal Care and Use Committee of the Development Center for Biotechnology (DCB) (Taipei, Taiwan) (Document number: IACUC#2023-R403-006).

[0149] Example 1. Analysis of the binding ability of transporter peptides to transferrin receptors In this example, the affinity of the transporter peptides of the present invention for the transferrin receptor was analyzed by fiber optic particle plasmon resonance (FOPPR). In this example, different amino acid positions of the transporter peptide were further replaced with different amino acids of the same nature, and the binding affinity of these mutant peptides to the transferrin receptor was analyzed.

[0150] Materials and Methods Each of the transporter peptides according to the present invention (SEQ ID NOs: 2 to 30) was dissolved in PBS buffer at a concentration of 10 μg / ml, pH 7.4. 80 μl of the solution was added dropwise to a standard sensor chip (NanoAu-MM, Instant NanoBiosensors, Taiwan) whose surface had been preactivated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) to conjugate the transporter peptide to the carboxyl groups on the surface of the sensor chip. Transferrin receptor (TfR protein, ACROBiosystems, product number: CD1-H5243, USA) was loaded onto the sensor chip at concentrations of 0.5 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL, respectively. The intermolecular interaction parameter K was measured by a FOPPR system detector (FOPPR system D200 (Id M3), Taiwan) based on the change in light intensity. D was calculated.

[0151] result The affinity of the transporter peptide of the present invention to the transferrin receptor is D The values ​​are shown as K values ​​(Kinetic data). D A higher value indicates a weaker bond between the two molecules; conversely, a higher KD A lower value indicates a stronger bond between the two molecules (K D (A value <300 nM indicates that the tested peptide has a strong binding affinity for TfR.) As shown in Table 2, each of the transporter peptides disclosed herein (SEQ ID NO: 2 to SEQ ID NO: 30) has a strong binding affinity to the transferrin receptor. These results demonstrate that any of the sites in the transporter peptide disclosed herein (SEQ ID NO: 1) can be substituted with several specific amino acids without impairing the binding affinity for TfR.

[0152] TIFF2025538660000003.tif209151

[0153] Example 2. In vitro transcytosynthesis analysis of transporter peptides In this example, the transport efficiency of the transporter peptides of the present invention across Caco-2 cell monolayers is measured to analyze the tissue barrier penetration effect of the transporter peptides of the present invention and their ability to induce transcytosheath development in the cells.

[0154] Materials and Methods cell culture 2x10 Caco-2 cells 4 individual cells / cm 2The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) in 24-well plates (24-transwell inserts, BD Falcon, USA, product number 353495) at a density of 100 μm. The cells were cultured for 21 days in DMEM containing 10% fetal bovine serum to form a cell monolayer. The transepithelial electrical resistance (TEER) of the cell monolayer was at least 500-600 Ω·cm. 2 to reach a monolayer acceptable for subsequent cell passage experiments.

[0155] High Performance Liquid Chromatography (HPLC) analysis HPLC analysis was performed using a Water ACQUITY Arc system. The column temperature of the C-18 column (Waters XSelect HSS T3 column) was maintained at 40 °C, and the mobile phase consisted of (A) 0.1% trifluoroacetic acid in water and (B) 100% methanol. The separation process followed a gradient process: 0 min - 10% (B), 6 min - 30% (B), 12 min - 50% (B), 18 min - 90% (B), and 23 min - 10% (B). The mobile phase flow rate was 1 mL / min, the sample injection volume was 20 μL, and the total run time was within 30 min.

[0156] In vitro transcytosheath analysis TEER value is 680 Ω·cm 2The transporter peptide of the present invention (PT-034, SEQ ID NO: 19) was placed at a concentration of 10 μM on top of a Caco-2 monolayer culture medium balanced with serum-free DMEM medium at 37°C. The upper and lower solutions were collected at 0, 0.25, 0.5, 1, 2, and 6 hours, respectively. The peptide in the sample solution was purified by solid-phase extraction (SPE) using C-18 pipette tips (Pierce, Product No. 87784, USA). Each sample was supplemented with PT-034 at a final concentration of 11.43 μM as an internal control. 300 μL of sample was adsorbed onto a C-18 pipette tip and eluted with 100 μL of methanol, followed by HPLC analysis. The transporter peptide content in each sample solution was calculated by calculating the HPLC peak area at a retention time of 16.9 minutes and establishing a standard curve. The transporter peptide concentrations in the upper and lower layers were calculated to determine the percentage of transporter peptide that crossed the Caco-2 cell monolayer.

[0157] result As shown in Figure 1, approximately 8%, 40%, 42%, and 56% of the transporter peptide (SEQ ID NO: 19) penetrated the Caco-2 cell monolayer at 0.25, 1, 2, and 6 hours, respectively. These results demonstrate that the transporter peptides of the present invention have the ability to transport across tissue barriers and can effectively induce the cells to form transcytosheaths.

[0158] Example 3. In vivo imaging system analysis of transporter peptides In this example, the transporter peptide according to the present invention is administered to mice, and the distribution of the transporter peptide in the mouse body is measured using an in vivo fluorescence imaging system (IVIS).

[0159] Materials and Methods Conjugation of peptides with fluorescent dyes 1 mg of each of the transporter peptides PT-001 (SEQ ID NO: 2), PT-025 (SEQ ID NO: 11), PT-034 (SEQ ID NO: 19), and PT-031 (SEQ ID NO: 17) was dissolved in 1 mL of buffer containing 50 mM sodium bicarbonate / bicarbonate salt to a final concentration of 1 mg / mL. 50 μL of 10 mg / mL fluorescent dye (VivoTag 680 XL, PerkinElmer, USA) was added to the 1 mL transporter peptide solution, and the mixture was incubated at room temperature for 1 hour, protected from light. The dye-labeled product was filtered through a 3 KDa centrifugal filter device (Amicon Ultra-0.5 Centrifugal filter device, Millipore, USA) and centrifuged at 14,000 x g for 15 minutes. The fluorescent dye-conjugated transporter peptides were diluted to a final volume of 0.2 mL with 1x PBS.

[0160] Animal experiments Seven- to eight-week-old BALB / c mice (BioLASCO Taiwan, Taiwan) were divided into a control group (n = 1) and an experimental group (n = 3). Control mice received 50 μL of fluorescent dye via tail vein injection. Experimental mice received 50 μL of the transporter peptide conjugated with the fluorescent dye via tail vein injection. Images of the mice were captured using an optical imaging device (U-OI, MlLabs, The Netherlands) for IVIS imaging analysis at 0.5, 1, 2, 4, 6, and 24 hours post-injection. Analysis was performed using an excitation wavelength of 631 nm, an emission wavelength of 710 nm, and a 10-second exposure time. The fluorescent signal in the mouse brain was then quantified.

[0161] result 3.1 Biodistribution of PT-034 As shown in Figure 2A, fluorescent signals accumulated in the brain and spine of experimental mice administered 10 mg / kg of the fluorescent dye-conjugated transporter peptide PT-034 (SEQ ID NO: 19) at 1, 2, 4, and 6 hours after injection. In contrast, no such fluorescent signal distribution was observed in the body of control mice (Dye). The fluorescent signals in the mouse brain were further quantified. As shown in Figure 2B, compared to the control group, a large amount of fluorescent signal was observed in the brain of experimental mice at 1 hour after injection, and the half-life of the fluorescence in the mouse brain was approximately 5 hours. The experimental results demonstrated that the transporter peptide of the present invention has the effect of targeting the brain and spine and is effective as a drug delivery system.

[0162] 3.2 Biodistribution of PT-001, PT-025, and PT-031 As shown in Figure 3A, fluorescent signals accumulated in the brain and spinal cord of experimental mice administered 1 mg / kg of fluorescent dye-conjugated transporter peptides PT-001 (SEQ ID NO: 2), PT-025 (SEQ ID NO: 11), and PT-031 (SEQ ID NO: 17) at 0.5, 1, 2, 4, and 6 hours after injection. The fluorescent signals in the mouse brain were further quantified. As shown in Figure 3B, a large amount of fluorescent signals was observed in the brain of the experimental mice at 0.5 hours after injection, and the half-life of the fluorescence in the mouse brain was approximately 5 hours. The experimental results demonstrated that peptides (SEQ ID NO: 2 to SEQ ID NO: 30) in which different amino acids were substituted at different amino acid positions in the transporter peptide (SEQ ID NO: 1) of the present invention have the effect of targeting the brain and spinal cord and are effective as drug delivery systems.

[0163] Example 4. Analysis of transport efficiency of transporter peptides in the brain In this example, the transport efficiency of the transporter peptide of the present invention in the brain is measured, thereby proving that the transporter peptide of the present invention has the effect of transporting the peptide across the blood-brain barrier.

[0164] Materials and Methods Mice (n=2) were administered 10 mg / kg of the transporter peptide of the present invention (PT-034, SEQ ID NO: 19) via tail vein injection. Brain tissue was collected 1 hour after injection and ground in 1.3 mL of 50 mM sodium bicarbonate / bicarbonate buffer. The brain tissue grinding solution was centrifuged at 3,000 rpm for 5 minutes, and the supernatant was collected and centrifuged at 14,000 rpm for 15 minutes. 0.4 mL of cold methanol was added to 0.1 mL of the brain tissue grinding supernatant, and the mixture was shaken for 10 minutes and immediately centrifuged at 14,000 rpm for 15 minutes. The brain tissue supernatant was then analyzed by HPLC. The HPLC analysis method was as described in Example 2. The HPLC peak area at the retention time of 16.9 minutes was calculated, and the transporter peptide content in each sample was calculated by establishing a standard curve.

[0165] result As shown in Table 3, the contents of the transporter peptide (PT-034) in the intact brain tissues of mice in both experimental groups (brain tissue-1 and brain tissue-2) were 25.5 μg and 25.9 μg, respectively. When converted to percent injected dose per gram of brain tissue (%ID / g of brain, injected dose / weight of brain tissue), these contents were 22.09% and 23.48%, respectively. The average efficiency of transporter peptide transport into the cerebrum was approximately 22.79%ID / g. These experimental results demonstrated that the transporter peptide of the present invention has the effect of transporting substances across the blood-brain barrier.

[0166] TIFF2025538660000004.tif52151

[0167] Example 5. In vivo imaging system analysis of transporter peptide conjugates (conjugation with antibodies) In this example, a transporter peptide conjugate was formed by conjugating a transporter peptide of the present invention with an antibody. The transporter peptide conjugate was then administered to mice, and the distribution of the transporter peptide conjugate in the mouse body was measured using an in vivo fluorescence imaging system (IVIS). In this example, the transporter peptide conjugate was administered by intravenous injection and intravenously, respectively, to examine the effects of different administration routes.

[0168] Materials and Methods Preparation of antibody conjugates with fluorescent dye-labeled transporter peptides An anti-Her-2 antibody (trastuzumab, Selleckchem, USA) was prepared at a concentration of 1 mg / mL in 100 mM carbonate / bicarbonate buffer. A crosslinker (bis(sulfosuccinimidyl) suberate, Thermo Scientific, USA) was dissolved in water to a concentration of 25 mM. 1 mL of the anti-Her-2 antibody, 11 μL of the crosslinker solution, and 8.4 μL of the transporter peptide of the present invention (concentration: 1 mg / mL) were mixed and reacted at room temperature for 30 minutes to carry out a covalent synthesis reaction. The reaction mixture was centrifuged through a 3 KDa filter at 14,000 × g for 15 minutes to obtain the transporter peptide conjugate (conjugated with the anti-Her-2 antibody). The volume of the product was adjusted to 1 mL with 50 mM carbonate buffer, and 10 μL of 10 mg / mL fluorescent dye (VivoTag 680 XL) was added. The mixture was left at room temperature for 1 hour, protected from light. The product was then centrifuged at 14,000 x g for 15 minutes using a 3 KDa filter. Finally, the total volume of the product was adjusted to 0.2 mL with 100 mM carbonate buffer. This product is a fluorescent dye-labeled transporter peptide conjugate (Ab-PT-D).

[0169] Preparation of fluorescent dye-labeled antibodies The anti-Her-2 antibody solution was added with 10 μL of a 10 mg / mL fluorescent dye (VivoTag 680 XL) and allowed to stand at room temperature for 1 hour, protected from light. The antibody was then labeled with the fluorescent dye (Ab-D) by centrifuging the solution at 14,000 x g for 15 minutes using a 3 KDa filter. The final volume of the antibody solution was adjusted to 0.2 mL with 50 mM carbonate buffer, and this was used as the negative control in this experiment.

[0170] Animal experiments Seven- to eight-week-old BALB / c mice (BioLASCO Taiwan) were administered the above fluorescent dye-labeled transporter peptide conjugates. After 0.5, 1, 2, 4, 6, and 24 hours, the mice were imaged using an optical imaging device (U-OI, M1Labs, The Netherlands) and subjected to IVIS imaging analysis. Analysis was performed using an excitation wavelength of 631 nm, an emission wavelength of 710 nm, and a 10-second exposure time. The fluorescent signal in the mouse brain was then quantified.

[0171] result 5.1 Intravenous injection The transporter peptide used in this experiment was PT-034 (SEQ ID NO: 19). Mice (n = 3) in the control group (Ab-D) were administered the fluorescent dye-labeled antibody at a dose of 10 mg / kg via tail vein injection. Mice (n = 3) in the experimental group (Ab-PT-D) were administered the fluorescent dye-labeled transporter peptide conjugate at a dose of 10 mg / kg via tail vein injection. As shown in Figure 4A, imaging results indicated that fluorescent signals were accumulated in the brain and spine of the experimental group (Ab-PT-D) administered with the fluorescent dye-labeled transporter peptide conjugate, and the distribution of the fluorescent signals was stronger than that of the control group. As shown in Figure 4B, the fluorescent signals in the mouse brain were further quantified in this example. Higher fluorescent signals were observed in the brain of the experimental group compared to the control group. This experiment demonstrated that the transporter peptide conjugate formed by conjugating an antibody to the transporter peptide of the present invention still has the effect of targeting the brain and spinal cord, and is therefore effective as a drug delivery system.

[0172] 5.2 Oral administration The transporter peptide used in this experiment was PT-034 (SEQ ID NO: 19). Mice in the intravenous injection group (n=2) were administered the fluorescent dye-labeled transporter peptide conjugate at a dose of 0.83 mg / kg via tail vein injection. Mice in the oral administration group (n=2) were orally administered the fluorescent dye-labeled transporter peptide conjugate at a dose of 0.83 mg / kg. As shown in Figure 5A, imaging results indicated that fluorescent signals were observed in the brain or spine of both the intravenous injection group and the oral administration group, which received the fluorescent dye-labeled transporter peptide conjugate. As shown in Figure 5B, clear fluorescent signals were detected in the brain of the oral administration group, demonstrating that the transporter peptide of the present invention was administered orally. As shown in Figure 5C, further quantification of the fluorescent signals in the mouse brains in this example indicated that fluorescent signals were observed in the brain of both the intravenous injection group and the oral administration group. The transporter peptides of the present invention have been administered orally or intravenously, both of which have been shown to have the effect of targeting the brain and to be effective as drug delivery systems.

[0173] The above results demonstrate that the transporter peptide conjugate of the present invention can maintain its efficacy whether administered intravenously or orally. Oral administration of the transporter peptide of the present invention allows the transporter peptide to bind to transferrin receptors present on cells in the gastrointestinal mucosal barrier, inducing cellular transcytosis, thereby transporting the peptide through the gastrointestinal mucosal barrier and targeting the brain after entering the body. The transporter peptide also crosses the blood-brain barrier via the same mechanism.

[0174] Example 6. In vivo imaging system analysis of transporter peptide conjugates (conjugation with lipid nanoparticles) In this example, a transporter peptide conjugate according to the present invention was conjugated to a lipid nanoparticle (LNP) to form a transporter peptide conjugate. The transporter peptide conjugate was then administered to mice, and the distribution of the transporter peptide conjugate in the mouse body was measured using an in vivo fluorescence imaging system (IVIS).

[0175] Materials and Methods Preparation of fluorescent dye-labeled lipid nanoparticles Lipid nanoparticles labeled with fluorescent dye (LNP-D) were prepared by mixing 2000 μg of lipid nanoparticles (LNP-102, ABP Biosciences, USA) dissolved in ethanol with 200 μg of fluorescent dye (VivoTag 680 XL) dissolved in 50 mM sodium acetate solution, gently stirring at room temperature for 30 minutes, and then dialysis against PBS for 24 hours in a 2K molecular weight cutoff dialysis cassette (Slide-A-Lyzer, ThermoFisher Scientific, USA).

[0176] Preparation of fluorescent dye-labeled transporter peptide-lipid nanoparticle conjugates A 0.1 mL portion of the transporter peptide of the present invention (1 mg / mL) was mixed with 5.8 μL of a crosslinker solution (EZ-Link TFP Ester-PEG4-DBCO, ThermoFisher Scientific, USA) (20 mg / mL) and reacted at room temperature for 1 hour. A total of 106 μL of this solution was added to 475 μL of the fluorescent dye-labeled lipid nanoparticle solution and reacted at room temperature for 1 hour to prepare a fluorescent dye-labeled transporter peptide / lipid nanoparticle conjugate (LNP-PT-D).

[0177] Animal experiments Seven- to eight-week-old BALB / c mice were divided into control and experimental groups. Control mice (n = 3) received 10 mg / kg of the fluorescent dye-labeled lipid nanoparticles (LNP-D) via tail vein injection. Experimental mice (n = 3) received 10 mg / kg of the fluorescent dye-labeled transporter peptide conjugate (LNP-PT-D) via tail vein injection. After 0.5, 1, 2, 4, 6, and 24 hours, images were captured using an optical imaging device (U-OI) and analyzed using IVIS imaging. Analysis was performed using an excitation wavelength of 631 nm, an emission wavelength of 710 nm, and a 10-second exposure time. The fluorescent signal in the mouse brain was quantified.

[0178] result The transporter peptide used in this experiment was PT-034 (SEQ ID NO: 19). As shown in Figure 6A, the imaging results indicated that the experimental group (LNP-PT-D) administered with the fluorescent dye-labeled transporter peptide conjugate had accumulated fluorescent signals in the brain and spinal cord, and had a stronger fluorescent signal distribution than the control group (LNP-D). As shown in Figure 6B, a higher fluorescent signal was observed in the brain of the experimental group (LNP-PT-D) compared to the control group (LNP-D). This experiment demonstrated that the transporter peptide conjugate formed by conjugating an antibody to the transporter peptide of the present invention still has the effect of targeting the brain and spinal cord, making it effective as a drug delivery system.

[0179] In summary, experiments have demonstrated that the transporter peptides of the present invention have binding affinity to the transferrin receptor. After binding to the transferrin receptor present on cells at the tissue barrier, the transporter peptides of the present invention induce cellular transcytosthesia, thereby transporting the transporter peptide and its conjugates through the tissue barrier. Experiments have demonstrated that the transporter peptides of the present invention (SEQ ID NO: 1) still maintain their binding affinity to the transferrin receptor after some amino acid sites are replaced with amino acids of the same nature. The transporter peptides of the present invention can be conjugated with an effector to form a transporter peptide conjugate. Experiments have demonstrated that the transporter peptide conjugates of the present invention maintain their binding affinity to the transferrin receptor. Furthermore, the transporter peptide conjugates of the present invention have the effect of transporting the effector through the tissue barrier, effectively resolving the problem of drug difficulty in crossing the blood-brain barrier. These conjugates have broad application value in the prevention and / or treatment of central nervous system diseases and contribute to the development of related clinical medical fields.

[0180] Of course, various changes and modifications can be made in the above-described embodiments of the invention without departing from the scope of the invention. Therefore, in order to promote the progress of science and useful arts, it is the intention to disclose the invention and to limit it only as set forth in the appended claims.

Claims

1. A transporter peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, which is capable of binding to the transferrin receptor (TfR).

2. 2. The transporter peptide of claim 1, wherein the amino acid sequence is selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 2 to SEQ ID NO: 30 and variant sequences that have at least 70% homology to any of the amino acid sequences set forth in SEQ ID NO: 2 to SEQ ID NO: 30 and are capable of binding to the transferrin receptor.

3. A nucleic acid encoding the transporter peptide of claim 1.

4. The nucleic acid of claim 3, which is a deoxyribonucleic acid or a ribonucleic acid.

5. A vector comprising the nucleic acid of claim 3.

6. The vector of claim 5, which is an adeno-associated virus vector.

7. The vector of claim 5 further comprising a nucleic acid encoding an effector.

8. The vector according to claim 7, wherein the effector is at least one selected from the group consisting of a peptide, a protein, an antibody, a virus particle, a liposome, an endosome, an exosome, a ligand, a eukaryotic cell, a prokaryotic cell, and a microsphere.

9. A recombinant transporter peptide conjugate expressed by the vector of claim 7.

10. A recombinant host cell comprising components selected from the group consisting of the peptide of claim 1, the nucleic acid of claim 3, and the vector of claim 5.

11. A transporter peptide conjugate comprising the transporter peptide of claim 1 and an effector, wherein the transporter peptide and the effector are conjugated in a covalent or non-covalent manner.

12. The transporter peptide conjugate of claim 11, wherein the effector is at least one selected from the group consisting of siRNA, shRNA, microRNA, double-stranded RNA, single-stranded RNA, DNA, oligonucleotide, aptamer, gene, peptide, protein, antibody, small chemical molecule, large chemical molecule, virus particle, liposome, endosome, exosome, nanoparticle, lipid nanoparticle, dendrimer, ligand, eukaryotic cell, prokaryotic cell, microsphere, nanogel, and bio-nanocapsule.

13. The transporter peptide of claim 1 ; The recombinant transporter peptide conjugate of claim 9; or The transporter peptide conjugate of claim 11. A composition comprising:

14. The composition of claim 13 further comprising a pharmaceutically acceptable carrier.

15. 10. Use of the transporter peptide of claim 1 in the preparation of a pharmaceutical composition for transporting an effector to a target, wherein the target is inside or outside the body.

16. The use according to claim 15, wherein the transporter peptide and the effector are conjugated in a covalent or non-covalent manner to form the transporter peptide conjugate according to claim 11.

17. The use of claim 15, wherein the transporter peptide and the effector form a recombinant transporter peptide conjugate of claim 9, such that nucleic acids encoding the transporter peptide and the effector are expressed.

18. 16. The use of claim 15, wherein the transporter peptide and the effector must cross a tissue barrier to reach the target, and the transporter peptide binds to a transferrin receptor on cells of the tissue barrier.

19. 19. The use of claim 18, wherein binding of the transporter peptide to the transferrin receptor induces transcytosheathment of the cell, thereby transporting the transporter peptide and the effector across the tissue barrier to reach the target.

20. 19. The use of claim 18, wherein the tissue barriers include the blood-brain barrier, the mucosal barrier, and the gastrointestinal barrier.

21. 16. The use of claim 15, wherein the target is a cell that expresses a transferrin receptor, and the transporter peptide transports the effector to the target by binding to the transferrin receptor expressed on the target cell.

22. 10. Use of the transporter peptide of claim 1 in the preparation of a pharmaceutical composition for transporting an effector across a tissue barrier in a subject.

23. The use according to claim 22, wherein the transporter peptide and the effector are conjugated in a covalent or non-covalent manner to form the transporter peptide conjugate according to claim 11.

24. 23. The use of claim 22, wherein the transporter peptide and the effector form a recombinant transporter peptide conjugate according to claim 9, such that nucleic acids encoding the transporter peptide and the effector are expressed.

25. 23. The use of claim 22, wherein the tissue barriers include the blood-brain barrier, the mucosal barrier, and the gastrointestinal barrier.

26. 23. The use of claim 22, wherein the cells of the tissue barrier have transferrin receptors on them.

27. 27. The use of claim 26, wherein the transporter peptide binds to the transferrin receptor, and binding of the transporter peptide to the transferrin receptor induces transcytosheathment of the cell, thereby transporting the transporter peptide and the effector across the tissue barrier.

28. Use of the transporter peptide of claim 1 in the preparation of a pharmaceutical composition for treating and / or preventing a central nervous system disease.

29. The use according to claim 28, wherein the transporter peptide and the effector are conjugated in a covalent or non-covalent manner to form the transporter peptide conjugate according to claim 11.

30. 29. The use of claim 28, wherein the transporter peptide and the effector form a recombinant transporter peptide conjugate according to claim 9, such that nucleic acids encoding the transporter peptide and the effector are expressed.

31. 31. The use according to claim 29 or 30, wherein the effector is a therapeutic agent for a central nervous system disease.

32. 31. The use according to claim 29 or 30, wherein the transporter peptide conjugate or the recombinant transporter peptide conjugate is administered to a subject in need thereof.

33. 29. The use of claim 28, wherein the transporter peptide binds to a transferrin receptor on cells of a tissue barrier in the subject's body, and the binding of the transporter peptide to the transferrin receptor induces transcytosheathment of the cells, thereby transporting the transporter peptide and the therapeutic agent for the central nervous system disease across the tissue barrier.

34. 34. The use of claim 33, wherein the tissue barriers include the blood-brain barrier, the mucosal barrier, and the gastrointestinal barrier.

35. 29. The use of claim 28, wherein the central nervous system disease comprises Alzheimer's disease, Parkinson's disease, cerebrovascular disease, vascular dementia, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, and spinal muscular atrophy.

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