Transport peptide and application thereof

Abstract

The present invention relates to a transporter peptide capable of binding to a transferrin receptor. The transporter peptide can be covalently or non-covalently conjugated with an effector to form a transporter peptide conjugate, or the transporter peptide and an effector form a recombinant transporter peptide conjugate in a manner that represents a nucleic acid encoding the transporter peptide and the effector. The transporter peptide and the recombinant transporter peptide transport the effector to a target by binding to a transferrin receptor. Binding of the transporter peptide to a transferrin receptor on a cell of a tissue barrier induces transendocytosis of the cell to transport the transporter peptide conjugate through the tissue barrier. The transport peptide can be used as a drug delivery system and can be used for related treatment of central nervous system (CNS) diseases.

Description

[0001] Cross - reference to related applications

[0002] This application claims the international priority and benefit of U.S. Provisional Patent Application No. 63 / 385,266, filed on November 29, 2022, the content of which is incorporated herein by reference into the present invention.

[0003] Submission of the sequence listing in ASCII text invention format

[0004] This application includes a sequence listing submitted electronically in XML format. The XML - formatted sequence listing includes a sequence list named "P23 - 0222PCT_Sequence_Listing.xml", created on November 6, 2023, with a size of 30,912 bytes. The sequence listing included in the XML - formatted sequence listing is part of the specification and is hereby incorporated by reference in its entirety into the present invention. Technical field

[0005] The present invention relates to transport peptides capable of crossing tissue barriers and methods of using these transport peptides to transport effectors across tissue barriers. Background art

[0006] The main function of tissue barriers is to restrict the passage of substances. They exist in various tissues and organs of living organisms to maintain the environmental stability of specific regions or protect important organs from external substances. For example, the blood - brain barrier (BBB) and the gastrointestinal mucosal barrier, etc.

[0007] The blood - brain barrier is located in the central nervous system and is composed of cerebral microvascular endothelial cells (BMECs). Its main function is to protect the brain from external substances and also control the passage of specific substances (such as oxygen and nutrients, etc.) to ensure the normal operation of the brain. However, in clinical treatment, the existence of the blood - brain barrier significantly restricts the entry of therapeutic drugs into the brain. Relevant clinical studies have pointed out that the proportion of neuro - drugs in the brain reaching the brain through the blood - brain barrier is less than 0.1%. Therefore, the drug delivery barrier caused by the blood - brain barrier has always been a long - standing problem in the related fields of the treatment of central nervous system (CNS) diseases. Summary of the invention

[0008] The present invention is at least partially based on the following discovery: A transport peptide comprising the amino acid sequence shown in SEQ ID NO:1 has a binding affinity for the transferrin receptor (TfR). These transport peptides can effectively bind to the transferrin receptor on the tissue barrier and induce transcytosis in cells, enabling these transport peptides to pass through the tissue barrier. In addition, these transport peptides can also effectively bind to the transferrin receptor on a target cell. In certain embodiments, the transport peptides of the present invention include, but are not limited to, any of the amino acid sequences shown in SEQ ID NO:2 to SEQ ID NO:30, and variant sequences having at least 70% homology with any of the amino acid sequences shown in SEQ ID NO:2 to SEQ ID NO:30 and capable of binding to the transferrin receptor.

[0009] In certain embodiments, the transport peptides of the present invention are further conjugated to an effector in a covalent or non-covalent manner to produce a transport peptide conjugate. In certain embodiments, the transport peptides of the present invention form a recombinant transport peptide conjugate by expressing a nucleic acid encoding the transport peptide and the effector. These transport peptides transport the effector to a target by binding to a transferrin receptor. Therefore, the transport peptides of the present invention can be used as a drug delivery system. In certain embodiments, the binding of these transport peptides to the transferrin receptor on the cells of a tissue barrier induces transcytosis of the cells, and transports the effector conjugated to the transport peptide through the tissue barrier. Through the transport peptides and transport methods of the present invention, an effector can be effectively transported across a tissue barrier, especially the blood-brain barrier, and delivered into the brain. Therefore, the transport peptides and transport methods of the present invention have broad clinical application value in the prevention and / or treatment of central nervous system diseases.

[0010] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the present invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the following embodiments.

[0011] Embodiment 1. A transport peptide composed of 10 amino acids has the following general sequence:

[0012] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0013] wherein the first amino acid site (X1) is an acidic amino acid or a polar uncharged amino acid;

[0014] the second amino acid site (X2) is a non-polar amino acid;

[0015] The 3rd amino acid site (X3) is any amino acid;

[0016] The 4th amino acid site (X4) is an acidic amino acid or a non-polar amino acid;

[0017] The 5th amino acid site (X5) is a non-polar amino acid;

[0018] The 6th amino acid site (X6) is any amino acid;

[0019] The 7th amino acid site (X7) is a basic amino acid;

[0020] The 8th amino acid site (X8) is an acidic amino acid or a basic amino acid;

[0021] The 9th amino acid site (X9) is a non-polar amino acid or a polar uncharged amino acid; and

[0022] The 10th amino acid site (X10) is an acidic amino acid or a polar uncharged amino acid.

[0023] Embodiment 2. A transport peptide, consisting of 10 amino acids, having the following general sequence:

[0024] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0025] Wherein,

[0026] The 1st amino acid site (X1) is Asp, Glu, Asn, Gln, Cys, Ser, Thr or Tyr;

[0027] The 2nd amino acid site (X2) is Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp or Met;

[0028] The 3rd amino acid site (X3) is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, His, Asp, Asn, Glu, Gln, Lys, Arg, Ser, Thr, Met, Cys or Pro;

[0029] The 4th amino acid site (X4) is Asp, Glu, Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp or Met;

[0030] The 5th amino acid site (X5) is Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp or Met);

[0031] The 6th amino acid site (X6) is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, His, Asp, Asn, Glu, Gln, Lys, Arg, Ser, Thr, Met, Cys or Pro;

[0032] The 7th amino acid site (X7) is Lys, His or Arg;

[0033] The 8th amino acid site (X8) is Asp, Glu, Lys, His or Arg;

[0034] The 9th amino acid site (X9) is Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp, Met, Asn, Gln, Cys, Ser, Thr or Tyr; and

[0035] The 10th amino acid site (X10) is Asp, Glu, Asn, Gln, Cys, Ser, Thr or Tyr.

[0036] Embodiment 3. A transit peptide, consisting of 10 amino acids, having the sequence shown in SEQ ID NO:1 as follows:

[0037] X1-Ile-X3-Val-Leu-X6-Lys-X8-X9-X10 (SEQ ID NO:1),

[0038] wherein, the 1st amino acid site (X1) is Asp, Glu, Asn, Gln, Cys, Ser, Thr or Tyr;

[0039] Ile at the 2nd amino acid site (X2) can be replaced by Gly, Ala, Leu, Val, Pro, Phe, Trp or Met;

[0040] The 3rd amino acid site (X3) is any amino acid;

[0041] Val at the 4th amino acid site (X4) can be replaced by Asp, Glu, Gly, Ala, Ile, Leu, Pro, Phe, Trp or Met;

[0042] Leu at the 5th amino acid site (X5) can be replaced by Gly, Ala, Ile, Val, Pro, Phe, Trp or Met;

[0043] The 6th amino acid site (X6) is any amino acid;

[0044] Lys at the 7th amino acid site (X7) can be replaced by His or Arg;

[0045] The 8th amino acid site (X8) is His, Arg, Lys, Asp or Glu;

[0046] The 9th amino acid site (X9) is Gly, Ala, Ile, Leu, Val, Pro, Phe, Trp, Met, Asn, Gln, Cys, Ser, Thr or Tyr; and

[0047] The 10th amino acid site (X10) is Asp, Glu, Asn, Gln, Cys, Ser, Thr or Tyr.

[0048] Embodiment 4. The transport peptide according to any one of Embodiments 1 to 3, characterized in that the transport peptide can bind to a transferrin receptor (TfR).

[0049] Embodiment 5. The transport peptide according to any one of Embodiments 1 to 4, characterized in that the amino acid sequence of the transport peptide is selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30 and variant sequences having at least 70% homology with any one of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30 and capable of binding to the transferrin receptor.

[0050] Embodiment 6. The transport peptide according to Embodiment 5, characterized in that the variant sequence has 70% homology with any one of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30.

[0051] Embodiment 7. The transport peptide according to Embodiment 5, characterized in that the variant sequence has 80% homology with any one of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30.

[0052] Embodiment 8. The transport peptide according to Embodiment 5, characterized in that the variant sequence has 90% homology with any one of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 30.

[0053] Embodiment 9. A nucleic acid encoding the transport peptide according to any one of Embodiments 1 to 8.

[0054] Embodiment 10. The nucleic acid according to Embodiment 9, characterized in that the nucleic acid is deoxyribonucleic acid (DNA).

[0055] Embodiment 11. The nucleic acid as described in Embodiment 9, characterized in that the nucleic acid is ribonucleic acid (RNA).

[0056] Embodiment 12. A vector, comprising the nucleic acid as described in any one of Embodiments 9 to 11.

[0057] Embodiment 13. The vector as described in Embodiment 12, characterized in that the vector is an adeno-associated virus vector.

[0058] Embodiment 14. The vector as described in Embodiment 12, further comprising a nucleic acid encoding an effector.

[0059] Embodiment 15. The vector as described in Embodiment 14, characterized in that the effector is selected from at least one of the group consisting of peptides, proteins, antibodies, virus particles, liposomes, endosomes, exosomes, ligands, eukaryotic cells, prokaryotic cells, and microspheres.

[0060] Embodiment 16. A recombinant transport peptide conjugate, expressed by the vector as described in Embodiment 14 or 15.

[0061] Embodiment 17. A recombinant host cell, comprising a component selected from the group consisting of: the peptide as described in any one of Embodiments 1 to 8, the nucleic acid as described in any one of Embodiments 9 to 11, and the vector as described in any one of Embodiments 12 to 15.

[0062] Embodiment 18. A transport peptide conjugate, composed of the transport peptide as described in any one of Embodiments 1 to 8 and an effector, characterized in that the transport peptide and the effector are conjugated in a covalent or non-covalent manner.

[0063] Embodiment 19. The transport peptide conjugate as described in Embodiment 18, characterized in that the effector is selected from at least one of the group consisting of siRNA, shRNA, microRNA, double-stranded RNA, single-stranded RNA, DNA, oligonucleotides, aptamers, genes, peptides, proteins, antibodies, small chemical molecules, large chemical molecules, virus particles, liposomes, endosomes, exosomes, nanoparticles, lipid nanoparticles, dendrimers, ligands, eukaryotic cells, prokaryotic cells, microspheres, nanogels, and biocompatible nanocapsules.

[0064] Embodiment 20. A composition, comprising:

[0065] a transport peptide as described in any one of Embodiments 1 to 8;

[0066] a recombinant transport peptide conjugate as described in Embodiment 16; or

[0067] A transit peptide conjugate as described in embodiment 18 or 19.

[0068] Embodiment 21. The composition as described in embodiment 20, further comprising a pharmaceutically acceptable carrier.

[0069] Embodiment 22. A method of transporting a composition as described in embodiment 20 or 21 to a target, comprising binding the transit peptide, the transit peptide on the recombinant transit peptide conjugate, or the transit peptide on the transit peptide conjugate to a transferrin receptor.

[0070] Embodiment 23. The method as described in embodiment 22, wherein the composition must reach the target through a tissue barrier, and the transferrin receptor is located on a cell of the tissue barrier.

[0071] Embodiment 24. The method as described in embodiment 22 or 23, wherein the binding of the transit peptide of the composition, the transit peptide on the transit peptide conjugate of the composition, or the transit peptide on the recombinant transit peptide conjugate of the composition to the transferrin receptor induces transcytosis of the cell, thereby transporting the composition through the tissue barrier to reach the target.

[0072] Embodiment 25. The method as described in any one of embodiments 22 to 24, wherein the tissue barrier comprises a Blood-Brain Barrier (BBB), a Mucosal Barrier, and a Gastrointestinal Barrier.

[0073] Embodiment 26. The method as described in any one of embodiments 22 to 25, wherein the target is a brain cell.

[0074] Embodiment 27. The method as described in any one of embodiments 22 to 26, wherein the tissue barrier is a Blood-Brain Barrier (BBB), and the target is a brain cell.

[0075] Embodiment 28. The method as described in embodiment 22, wherein the target is a cell expressing the transferrin receptor, and the transferrin receptor is located on the target.

[0076] Embodiment 29. The method as described in embodiment 22 or 28, wherein the target is a cancer cell.

[0077] Embodiment 30. The method according to Embodiment 29, wherein the cancer is a hepatocellular carcinoma, breast cancer, lung cancer, colon cancer, brain cancer, glioma, prostate cancer, ovarian cancer, or leukemia.

[0078] Embodiment 31. The method according to Embodiment 22 or 29, wherein the target is a tissue cell.

[0079] Embodiment 32. The method according to Embodiment 31, wherein the tissue is skin, tonsil, tongue, oesophagus, cervix, kidney, placenta, pancreas, testis, anterior pituitary, stomach, breast, or liver.

[0080] Embodiment 33. The method according to any one of Embodiments 22 to 32, wherein the target is located in vivo or in vitro.

[0081] Embodiment 34. A method for transporting an effector through a tissue barrier of a subject, comprising: administering to the subject a recombinant transport peptide conjugate as described in Embodiment 16 or a transport peptide conjugate as described in Embodiment 18 or 19.

[0082] Embodiment 35. The method according to Embodiment 34, wherein the recombinant transport peptide conjugate or the transport peptide conjugate comprises the effector.

[0083] Embodiment 36. The method according to Embodiment 34 or 35, wherein the tissue barrier comprises a Blood-Brain Barrier (BBB), a Mucosal Barrier, and a Gastrointestinal Barrier.

[0084] Embodiment 37. The method according to any one of Embodiments 34 to 36, wherein a transferrin receptor is present on a cell of the tissue barrier.

[0085] Embodiment 38. The method according to any one of Embodiments 34 to 37, wherein the transport peptide binds to the transferrin receptor, and the binding of the transport peptide to the transferrin receptor induces transcytosis of the cell, thereby transporting the transport peptide and the effector through the tissue barrier.

[0086] Embodiment 39. The method according to any one of Embodiments 34 to 38, wherein the administration mode includes intradermal, intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal, intralung, and transdermal delivery.

[0087] Embodiment 40. The method according to any one of Embodiments 34 to 39, wherein the effector included in the recombinant transport peptide conjugate is selected from at least one of the group consisting of peptides, proteins, antibodies, virus particles, liposomes, endosomes, exosomes, ligands, eukaryotic cells, prokaryotic cells, and microspheres.

[0088] Embodiment 41. The method according to any one of Embodiments 34 to 39, wherein the effector included in the transport peptide conjugate is selected from at least one of the group consisting of siRNA, shRNA, microRNA, double-stranded RNA, single-stranded RNA, DNA, oligonucleotides, aptamers, genes, peptides, proteins, antibodies, small chemical molecules, large chemical molecules, virus particles, liposomes, endosomes, exosomes, nanoparticles, lipid nanoparticles, dendrimers, ligands, eukaryotic cells, prokaryotic cells, microspheres, nanogels, and bio-nanocapsules.

[0089] Embodiment 42. The method according to any one of Embodiments 34 to 41, wherein the subject is a mammal.

[0090] Embodiment 43. The method according to any one of Embodiments 34 to 42, wherein the subject is a rodent or a human subject.

[0091] Embodiment 44. A method for treating and / or preventing a Central Nervous System (CNS) disease, comprising: administering to a subject in need thereof a pharmaceutically effective amount of a recombinant transport peptide conjugate as described in Embodiment 16 or a transport peptide conjugate as described in Embodiment 18 or 19.

[0092] Embodiment 45. The method according to Embodiment 44, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0093] Embodiment 46. The method according to Embodiment 44 or 45, characterized in that the recombinant transport peptide conjugate or the effector comprised by the transport peptide conjugate is a therapeutic agent for a central nervous system disease.

[0094] Embodiment 47. The method according to any one of Embodiments 44 to 46, characterized in that the transport peptide on the recombinant transport peptide conjugate or the transport peptide on the transport peptide conjugate binds to a transferrin receptor on a cell of a tissue barrier in the subject, and the binding of the transport peptide to the transferrin receptor induces transcytosis of the cell, thereby transporting the transport peptide and the therapeutic agent for the central nervous system disease through the tissue barrier.

[0095] Embodiment 48. The method according to any one of Embodiments 44 to 47, characterized in that the tissue barrier comprises a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier.

[0096] Embodiment 49. The method according to any one of Embodiments 44 to 48, characterized in that the subject is a mammal.

[0097] Embodiment 50. The method according to any one of Embodiments 44 to 49, characterized in that the subject is a rodent or a human subject.

[0098] Embodiment 51. The method according to any one of Embodiments 44 to 50, characterized in that the administration mode comprises intradermal, intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal, intrapulmonary, and transdermal delivery.

[0099] Embodiment 52. The method according to any one of Embodiments 44 to 51, characterized in that the central nervous system diseases include 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 chorea, and Spinal Muscular Atrophy (SMA).

[0100] Embodiment 53. Use of a transport peptide according to any one of Embodiments 1 to 8 for transporting an effector to a target.

[0101] Embodiment 54. Use of a transport peptide according to any one of Embodiments 1 to 8 for preparing a pharmaceutical composition for transporting an effector to a target.

[0102] Embodiment 55. The use according to Embodiment 53 or 54, characterized in that the transport peptide is conjugated to the effector in a covalent or non-covalent manner to form a transport peptide conjugate as described in Embodiment 18 or 19.

[0103] Embodiment 56. The use according to Embodiment 53 or 54, characterized in that the transport peptide and the effector form a recombinant transport peptide conjugate as described in Embodiment 16 by expressing a nucleic acid encoding the transport peptide and the effector.

[0104] Embodiment 57. The use according to any one of Embodiments 53 to 56, characterized in that the transport peptide and the effector must reach the target through a tissue barrier, and the transport peptide binds to a transferrin receptor on a cell of the tissue barrier.

[0105] Use according to embodiment 58, characterized in that the binding of the transport peptide to the transferrin receptor induces transcytosis of the cell, and the transport peptide and the effector are transported through the tissue barrier to reach the target.

[0106] Embodiment 59. The use according to embodiment 57 or 58, characterized in that the tissue barrier includes a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier.

[0107] Embodiment 60. The use according to any one of embodiments 53 to 56, characterized in that the target is a cell expressing a transferrin receptor, and the transport peptide transports the effector to the target by binding to the transferrin receptor expressed on the target cell.

[0108] Embodiment 61. The use according to embodiment 60, characterized in that the target is a cancer cell or a tissue cell.

[0109] Embodiment 62. The use according to embodiment 61, characterized in that the cancer is a liver cancer, breast cancer, lung cancer, rectal cancer, brain cancer, glioma, prostate cancer, ovarian cancer, leukemia.

[0110] Embodiment 63. The use according to embodiment 61, characterized in that the tissue is skin, tonsil, tongue, esophagus, cervix, kidney, placenta, pancreas, testis, anterior pituitary, stomach, breast, liver.

[0111] Embodiment 64. The use according to any one of embodiments 53 to 63, characterized in that the target is located in vivo or in vitro.

[0112] Embodiment 65. Use of a transport peptide according to any one of embodiments 1 to 8 in transporting an effector through a tissue barrier of a subject.

[0113] Embodiment 66. Use of a transport peptide according to any one of embodiments 1 to 8 in the preparation of a pharmaceutical composition for transporting an effector through a tissue barrier of a subject.

[0114] Embodiment 67. The use according to embodiment 65 or 66, characterized in that the transport peptide and the effector are conjugated covalently or non-covalently to form a transport peptide conjugate as described in embodiment 18 or 19.

[0115] Embodiment 68. The use according to embodiment 65 or 66, characterized in that the transport peptide and the effector form a recombinant transport peptide conjugate as described in embodiment 16 by expressing a nucleic acid encoding the transport peptide and the effector.

[0116] Use according to any one of embodiments 65 to 68, characterized in that the tissue barrier comprises a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier.

[0117] Use according to any one of embodiments 65 to 69, characterized in that a transferrin receptor is present on a cell of the tissue barrier.

[0118] Use according to any one of embodiments 65 to 70, characterized in that the transport peptide binds to the transferrin receptor, and the binding of the transport peptide to the transferrin receptor induces transcytosis of the cell, thereby transporting the transport peptide and the effector through the tissue barrier.

[0119] Use of a transport peptide according to any one of embodiments 1 to 8 in the treatment and / or prevention of central nervous system diseases.

[0120] Use of a transport peptide according to any one of embodiments 1 to 8 in the preparation of a pharmaceutical composition for the treatment and / or prevention of central nervous system diseases.

[0121] Use according to embodiment 72 or 73, characterized in that the transport peptide is conjugated to an effector in a covalent or non-covalent manner to form a transport peptide conjugate according to embodiment 18 or 19.

[0122] Use according to embodiment 72 or 73, characterized in that the transport peptide and the effector form a recombinant transport peptide conjugate according to embodiment 16 by expressing a nucleic acid encoding the transport peptide and the effector.

[0123] Use according to embodiment 74 or 75, characterized in that the effector is a therapeutic agent for central nervous system diseases.

[0124] Use according to any one of embodiments 74 to 76, characterized in that the transport peptide conjugate or the recombinant transport peptide conjugate is administered to a subject in need thereof.

[0125] Use according to embodiment 77, characterized in that the transport peptide binds to a transferrin receptor on a cell of a tissue barrier in the subject's body, and the binding of the transport peptide to the transferrin receptor induces transcytosis of the cell, thereby transporting the transport peptide and the therapeutic agent for central nervous system diseases through the tissue barrier.

[0126] Use according to embodiment 78, characterized in that the tissue barrier comprises a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier.

[0127] Use according to any one of embodiments 72 to 79, characterized in that the central nervous system diseases include Alzheimer's disease, Parkinson's disease, cerebrovascular accident, vascular-related dementia, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's chorea, and spinal muscular atrophy.

[0128] Use according to any one of embodiments 53 to 55, 57 to 67, 69 to 74, 76 to 80, characterized in that the effector is selected from at least one 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.

[0129] Use according to any one of embodiments 53 to 54, 56 to 66, 68 to 73, 75 to 80, characterized in that the effector is selected from at least one of the group consisting of peptide, protein, antibody, virus particle, liposome, endosome, exosome, ligand, eukaryotic cell, prokaryotic cell, and microsphere.

[0130] Use according to any one of embodiments 65 to 82, characterized in that the subject is a mammal.

[0131] Use according to any one of embodiments 65 to 83, characterized in that the subject is a rodent or a human subject.

[0132] From the following description of the preferred embodiments in conjunction with the accompanying drawings, these and other aspects will become apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] The drawings illustrate one or more embodiments of the present invention and are used, together with the written description, to explain the principles of the present invention. Wherever possible, the same reference numerals are used throughout the drawings to refer to the same or similar components in the embodiments.

[0134] Figure 1 The figure shows the experimental results of the transport efficiency of the transport peptide (PT-034, SEQ ID NO: 19) on Caco-2 monolayer cells.

[0135] Figure 2A In vivo fluorescence imaging pictures of mice after administration of the transport peptide (PT-034, SEQ ID NO: 19) of the present invention at 1, 2, 4, 6, and 24 hours respectively; Figure 2B It is a quantitative graph of the fluorescence intensity of mouse brain tissue.

[0136] Figure 3A In vivo fluorescence imaging pictures of mice after administration of the transport peptide (PT-001, SEQ ID NO: 2, PT-025, SEQ ID NO: 11, or PT-031, SEQ ID NO: 17) of the present invention at 0.5, 1, 2, 4, 6, and 24 hours respectively; Figure 3B It is a quantitative graph of the fluorescence intensity of mouse brain tissue.

[0137] Figure 4A In vivo fluorescence imaging pictures of mice after administration of the transport peptide conjugate (conjugate of PT-034 and antibody) of the present invention at 0.5, 1, 2, 4, 6, and 24 hours respectively; Figure 4B It is a quantitative graph of the fluorescence intensity of mouse brain tissue.

[0138] Figure 5A In vivo fluorescence imaging pictures of mice after intravenous injection or oral administration of the transport peptide conjugate (conjugate of PT-034 and antibody) of the present invention at 0.5, 1, 2, 4, 6, and 24 hours respectively; Figure 5B It is a fluorescence imaging picture of mouse brain; Figure 5C It is a quantitative graph of the fluorescence intensity of mouse brain tissue.

[0139] Figure 6A In vivo fluorescence imaging pictures of mice after administration of the transport peptide conjugate (conjugate of PT-034 and lipid nanoparticles) of the present invention at 0.5, 1, 2, 4, 6, and 24 hours respectively; Figure 6B It is a quantitative graph of the fluorescence intensity of mouse brain tissue. Detailed implementation mode

[0140] The present invention is at least partially based on the following findings: The transport peptides of the present invention can effectively bind to the transferrin receptor on a cell. In certain embodiments, the transport peptides of the present invention can be conjugated to a substance in a covalent or non-covalent manner to form a transport peptide conjugate. In certain embodiments, the transport peptides of the present invention and an effector form a recombinant transport peptide conjugate by expressing a nucleic acid encoding the transport peptide and the effector. In the case where the cell is a cell on a tissue barrier, the binding of the transport peptide of the present invention to the transferrin receptor induces the cell to undergo transcytosis, so that the substance conjugated to the transport peptide of the present invention can pass through the tissue barrier. In the case where the cell is a target cell, the binding of the transport peptide of the present invention to the transferrin receptor can transport the conjugated substance to the target cell. Therefore, the transport peptides of the present invention can serve as a drug delivery system. In particular, the transport peptides of the present invention can be transported through a tissue barrier, particularly the blood-brain barrier, by transcytosis, and deliver the substance conjugated to the transport peptide of the present invention into the brain. Therefore, the transport peptides of the present invention can be used for the prevention and / or treatment of central nervous system diseases.

[0141] Specifically, the present invention provides a transport peptide composed of 10 amino acids, having the following general formula:

[0142] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0143] Wherein,

[0144] The first amino acid site (X1) is an acidic amino acid (Asp or Glu) or a polar uncharged amino acid (Asn, Gln, Cys, Ser, Thr or Tyr);

[0145] The second amino acid site (X2) is a non-polar amino acid (Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp or Met);

[0146] The third amino acid site (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 site (X4) is an acidic amino acid (Asp or Glu) or a non-polar amino acid (Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp or Met);

[0147] The fifth amino acid site (X5) is a non-polar amino acid (Ile, Gly, Ala, Leu, Val, Pro, Phe, Trp or Met);

[0148] The 6th amino acid site (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 7th amino acid site (X7) is a basic amino acid (Lys, His or Arg);

[0149] The 8th amino acid site (X8) is an acidic amino acid (Asp or Glu) or a basic amino acid (Lys, His or Arg);

[0150] The 9th amino acid site (X9) is a non-polar 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 site (X10) is an acidic amino acid (Asp or Glu) or a polar uncharged amino acid (Asn, Gln, Cys, Ser, Thr or Tyr).

[0151] Preferably and more specifically, the transport peptide provided by the present invention has the sequence shown in the following SEQ ID NO:1:

[0152] X1-Ile-X3-Val-Leu-X6-Lys-X8-X9-X10 (SEQ ID NO:1),

[0153] wherein,

[0154] The 1st amino acid site (X1) is Asp, Glu, Asn, Gln, Cys, Ser, Thr or Tyr;

[0155] Ile at the 2nd amino acid site (X2) can be replaced by Gly, Ala, Leu, Val, Pro, Phe, Trp or Met;

[0156] The 3rd amino acid site (X3) is any amino acid;

[0157] Val at the 4th amino acid site (X4) can be replaced by Asp, Glu, Gly, Ala, Ile, Leu, Pro, Phe, Trp or Met;

[0158] Leu at the 5th amino acid site (X5) can be replaced by Gly, Ala, Ile, Val, Pro, Phe, Trp or Met;

[0159] The 6th amino acid site (X6) is any amino acid;

[0160] The Lys at the 7th amino acid position (X7) can be replaced with His or Arg;

[0161] The 8th amino acid position (X8) is His, Arg, Lys, Asp or Glu;

[0162] The 9th amino acid position (X9) is Gly, Ala, Ile, Leu, Val, Pro, Phe, Trp, Met, Asn, Gln, Cys, Ser, Thr or Tyr; and

[0163] The 10th amino acid position (X10) is Asp, Glu, Asn, Gln, Cys, Ser, Thr or Tyr.

[0164] Preferably and in certain embodiments, the amino acid sequence shown in SEQ ID NO:1 includes, but is not limited to, any of the amino acid sequences shown in SEQ ID NO:2 to SEQ ID NO:30 and variant sequences having at least 70% homology with any of the amino acid sequences shown in SEQ ID NO:2 to SEQ ID NO:30 and capable of binding to a transferrin receptor. Preferably and in certain embodiments, the variant sequence has at least 70% homology with any of the amino acid sequences shown in SEQ ID NO:2 to SEQ ID NO:30. Preferably and in certain embodiments, the variant sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homology with any of the amino acid sequences shown in SEQ ID NO:2 to 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.

[0165] To determine the percentage identity between two sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of the first amino acid sequence for optimal alignment with the second amino acid sequence). When calculating the percentage identity, exact matches are typically counted. Mathematical algorithms known in the art, such as the BLAST and Gapped BLAST programs, the NBLAST and XBLAST programs, or the ALIGN program, can be used to determine the percentage homology or identity between two sequences.

[0166] Table 1. Codes and sequence list of transport peptides (SEQ ID NO:2 to SEQ ID NO:30)

[0167]

[0168]

[0169] Preferably and in certain embodiments, the amino acid sequence as set forth in SEQ ID NO:1 has a binding affinity for the transferrin receptor.

[0170] As used herein, the term "peptide" refers to a molecular chain of amino acids, including L-type and D-type. If desired, the amino acids can be modified in vivo or in vitro, for example, by mannose glycosylation, glycosylation, amidation (especially C-terminal amide), carboxylation, or phosphorylation, provided that these modifications must maintain the biological activity of the original molecule. In addition, the peptide can be part of a chimeric protein. As used in the present invention, the term "transport peptide" is described from the perspective of the application of peptides and generally refers to a peptide that can bind to a specific receptor to induce a transport effect and is also a target peptide.

[0171] Functional derivatives of the peptide are also included in the present invention. Functional derivatives are intended to include peptides having one or more different amino acids throughout the sequence, which have deletions, substitutions, inversions, or additions. Amino acid substitutions that are expected to substantially not alter the biological and immunological activities have been described. Amino acid replacements between related amino acids or replacements that often occur in evolution include Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, and Ile / Val, etc.

[0172] The peptides according to the present invention can be produced by synthetic or recombinant DNA techniques. Methods for producing synthetic peptides are known in the art.

[0173] As used in the present invention, the nomenclature for describing the peptides of the present invention follows the conventional practice, where the amine group (N-terminal) and / or 5' is on the left, and the carboxyl group (C-terminal) and / or 3' is on the right. As used in the present invention, the term "amino acid site" refers to the amino acid at a specific position in the peptide, calculated from the N-terminal of the peptide. For example, the first amino acid site (X1) refers to the position of the first amino acid counted from the N-terminal of the peptide.

[0174] As used in the present invention, the term "any amino acid" refers to any one of the 20 basic amino acids. As used in the present invention, the term "basic amino acid" includes the following 20 amino acids: glycine (Glycine, Gly, G), alanine (Alanine, Ala, A), valine (Valine, Val, V), leucine (Leucine, Leu, L), isoleucine (Isoleucine, Ile, I), phenylalanine (Phenylalanine, Phe, F), tyrosine (Tyrosine, Tyr, Y), tryptophan (Tryptophan, Trp, W), histidine (Histidine, His, H), aspartic acid (Aspartic acid, Asp, D), asparagine (Asparagine, Asn, N), glutamic acid (Glutamic acid, Glu, E), glutamine (Glutamine, Gln, Q), lysine (Lysine, Lys, K), arginine (Arginine, Arg, R), serine (Serine, Ser, S), threonine (Threonine, Thr, T), methionine (Methionine, Met, M), cysteine (Cysteine, Cys, C), and proline (Proline, Pro, P). The symbols representing amino acids in the present invention are the same as the amino acid abbreviations used by those skilled in the art. Unless otherwise defined, as used in the present invention, 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. When the amino acid is not specifically indicated as a dextrorotatory or levorotatory amino acid, 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 specific isomer.

[0175] As used in the present invention, the term "transferrin receptor" (TfR) refers to a type II transmembrane glycoprotein with a molecular weight of 90 kDa and is found to form a homodimer (180 kDa) through disulfide bonds on the cell surface where it is located. The protein sequence of the transferrin receptor can be referred to the protein number P02786 in the Uniprot website. The extracellular domain of each transferrin receptor consists of three domains, including the apical domain (amino acid residues 189 - 383), the protease-like domain (amino acid residues 122 - 188 and 384 - 606), and the helical domain (amino acid residues 607 - 760). The region where transferrin binds is mainly located in the helical domain and a small part of the protease-like domain on the surface of the transferrin receptor. The main function of the transferrin receptor is to bind to transferrin (Fe-Transferrin, Tf, a protein that carries iron in the blood) and promote the uptake of iron by cells. The 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, the gastrointestinal barrier, and the mucosal barrier, etc. In addition, transferrin receptors are found to have a relatively high expression level in rapidly proliferating cells (such as cancer cells), for example, cells associated with liver cancer, breast cancer, lung cancer, rectal cancer, brain cancer, glioma, prostate cancer, ovarian cancer, and leukemia. Transferrin receptors are also found to be expressed in some tissue cells, and these tissues include, but are not limited to, skin, tonsil, tongue, esophagus, cervix, kidney, placenta, pancreas, testis, anterior pituitary gland, stomach, breast, and liver.

[0176] The present invention also provides a nucleic acid encoding the transport peptide as described in the present invention. Preferably and in certain embodiments, the nucleic acid is deoxyribonucleic acid (DNA). Preferably and in certain embodiments, the nucleic acid is ribonucleic acid (RNA).

[0177] As used herein, the term "nucleotide" refers to a monomer that includes a nitrogenous base attached to a sugar phosphate, where the sugar phosphate includes a sugar such as ribose or 2'-deoxyribose, attached to one or more phosphate groups. "Polynucleotide" and "nucleic acid" mean polymers that include more than one nucleotide monomer, where the monomers are typically linked by sugar-phosphate bonds of a sugar-phosphate backbone. A polynucleotide need not include only one type of nucleotide monomer. For example, the nucleotides that make up a given polynucleotide can be ribonucleotides only, 2'-oxynucleotides only, 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 that include one or more non-naturally occurring monomers. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), where "U" replaces "T". The term "cDNA" means a DNA that is complementary or identical to an mRNA, whether in single-stranded or double-stranded form, but where "T" replaces "U". The term "recombinant nucleic acid" means a polynucleotide or nucleic acid having sequences that are not naturally joined together. A recombinant nucleic acid can exist in the form of a vector.

[0178] The nucleic acid encoding any of the transport peptides of the present invention is derived from the amino acid sequence of the transport peptide of the present invention. By replacing each amino acid in the amino acid sequence of the transport peptide of the present invention with the nucleotide sequence encoding the amino acid listed in the genetic code table (including various degenerate codons, or synonymous codons), the nucleic acid sequence provided by the present invention can be obtained. For example, proline in the amino acid sequence of the transport peptide of the present invention can be encoded by nucleotide sequences such as CCA, CCC, CCG, CCT, etc.

[0179] The present invention also provides a vector that includes the nucleic acid encoding the transport peptide as described in the present invention. Preferably and in certain embodiments, the vector is an adeno-associated virus vector. Preferably and in certain embodiments, the vector further includes a nucleic acid encoding an effector. More preferably and in certain preferred embodiments, the effector is selected from at least one of the group consisting of peptides, proteins, antibodies, virus particles, liposomes, endosomes, exosomes, ligands, eukaryotic cells, prokaryotic cells, and microspheres.

[0180] As used in the present invention, the term "vector" refers to a DNA molecule that serves as a vehicle to carry foreign DNA into a host organism (usually a bacterium, yeast, or mammalian cell). Examples of vectors include, but are not limited to, plasmids, phages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), cosmids, shuttle vectors, expression vectors, retroviral vectors, and adenoviral vectors.

[0181] As used in the present invention, the term "adeno-associated virus (AAV)" refers to a small, non-enveloped virus belonging to the family Parvoviridae and the genus Dependoparvovirus. AAV does not replicate in infected cells, is not associated with any known human diseases, and is considered relatively safe for use as a gene delivery vector. As used in the present invention, the term "adeno-associated virus vector" refers to a vector derived by removing most of the viral genes of wild-type adeno-associated virus and replacing these genes with a target gene. AAV vectors are used to introduce specific genes or genetic material into target cells or tissues and are commonly used in gene therapy and molecular biology research.

[0182] As used in the present invention, the term "effector" generally refers to any molecule that has a function on a target within a tissue barrier, where the target generally refers to a target cell or an extracellular molecule.

[0183] As used herein, the term "antibody" refers to one or a group of polypeptides that include at least one binding domain formed by the folding of a polypeptide chain having a three-dimensional binding space with an inner surface shape and charge distribution complementary to the epitope of an antigen. Antibodies typically have a tetrameric form, including two pairs of identical polypeptide chains, each pair having one "light" chain and one "heavy" chain. The variable regions of each light / heavy chain pair form an antibody binding site. Antibodies can be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and antibodies and fragments that can be labeled in soluble or bound forms, including epitope-binding fragments, variants, or derivatives alone or in combination with other amino acid sequences. Antibodies can be from any species. The term antibody also includes binding fragments, including, but not limited to, Fv, Fab, Fab', F(ab')2, single stranded antibody (svFC), Diabody, and disulphide-linked variable region (dsFv). Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen-binding site. Antibody fragments can be fused or not fused to another immunoglobulin domain, including, but not limited to, an Fc region or a fragment thereof. Those skilled in the art will further understand that other fusion products can be produced, including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions.

[0184] The present invention also provides a recombinant transit peptide conjugate expressed by the vector of the present invention.

[0185] The present invention also provides a recombinant host cell comprising a component selected from one of the following: the peptide of the present invention, the nucleic acid of the present invention, and the vector of the present invention.

[0186] The present invention also provides a transport peptide conjugate, which is composed of any one of the transport peptides of the present invention and an effector conjugated to the transport peptide in a covalent or non-covalent manner. Preferably and in certain embodiments, the effector is selected from at least one of 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 biocompatible nanocapsule.

[0187] The present invention also provides a composition, comprising at least one of the following: the transport peptide of the present invention, the recombinant transport peptide conjugate of the present invention, and the transport peptide conjugate of the present invention. Preferably and in certain embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0188] As used in the present invention, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and other excipients or additives that are physiologically compatible. In certain embodiments, the carrier is suitable for intranasal, intravenous, intramuscular, intradermal, subcutaneous, parenteral, oral, transmucosal or transdermal administration. Depending on the route of administration, the active compound can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The use of such media and reagents for pharmaceutical active substances is known in the art.

[0189] The present invention also provides a method for transporting the composition of the present invention to a target, comprising binding the transport peptide, the transport peptide on the recombinant transport peptide conjugate, or the transport peptide on the transport peptide conjugate to a transferrin receptor. The present invention also provides the use of any one of the transport peptides of the present invention in transporting an effector to a target. The present invention also provides the use of any one of the transport peptides of the present invention in the preparation of a pharmaceutical composition for transporting an effector to a target. The target is located in vivo or in vitro.

[0190] Preferably and in certain embodiments, the composition must reach the target through a tissue barrier, and the transferrin receptor is located on a cell of the tissue barrier. Preferably and in certain embodiments, the transferrin of the composition, the transferrin on the transferrin conjugate of the composition, or the transferrin on the recombinant transferrin conjugate of the composition binds to the transferrin receptor to induce transcytosis of the cell, thereby transporting the composition through the tissue barrier to reach the target. Preferably and in certain embodiments, the tissue barrier includes a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier. Preferably and in certain embodiments, the target is a brain cell. More preferably and in certain preferred embodiments, the tissue barrier is a blood-brain barrier and the target is a brain cell.

[0191] Preferably and in certain embodiments, the target is a cell expressing the transferrin receptor, and the transferrin receptor is located on the target. Preferably and in certain embodiments, the target is a cancer cell. More preferably and in certain preferred embodiments, the cancer is a liver cancer, breast cancer, lung cancer, rectal cancer, brain cancer, glioma, prostate cancer, ovarian cancer, or blood cancer. Preferably and in certain embodiments, the target is a tissue cell. More preferably and in certain preferred embodiments, the tissue is skin, tonsil, tongue, esophagus, cervix, kidney, placenta, pancreas, testis, anterior pituitary, stomach, breast, or liver.

[0192] The present invention also provides a method for transporting an effector through a tissue barrier of a subject, comprising: administering to the subject a recombinant transferrin conjugate or a transferrin conjugate of the present invention. Use of a transferrin of the present invention for transporting an effector through a tissue barrier of a subject. Use of a transferrin of the present invention for preparing a pharmaceutical composition for transporting an effector through a tissue barrier of a subject.

[0193] Preferably and in certain embodiments, the recombinant transport peptide conjugate or the transport peptide conjugate comprises the effector. Preferably and in certain embodiments, the tissue barrier comprises a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier. Preferably and in certain embodiments, a transferrin receptor is present on a cell of the tissue barrier. Preferably and in certain embodiments, the transport peptide binds to the transferrin receptor, and the binding of the transport peptide to the transferrin receptor induces transcytosis of the cell to transport the transport peptide and the effector across the tissue barrier. Preferably and in certain embodiments, the mode of administration comprises intradermal, intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal, intrapulmonary, and transdermal delivery. Preferably and in certain embodiments, the effector comprised in the recombinant transport peptide conjugate is selected from at least one of 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. Preferably and in certain embodiments, the effector comprised in the transport peptide conjugate is selected from at least one of 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 biocompatible nanocapsule. Preferably and in certain embodiments, the subject is a mammalian animal. More preferably and in certain preferred embodiments, the subject is a rodent or a human subject.

[0194] The present invention also provides a method for treating and / or preventing a central nervous system (CNS) disease, comprising: administering to a subject in need thereof a pharmaceutically effective amount of the recombinant transport peptide conjugate of the present invention or the transport peptide conjugate of the present invention. The present invention also provides the use of the transport peptide of the present invention in treating and / or preventing a central nervous system disease. The present invention also provides the use of the transport peptide of the present invention in preparing a pharmaceutical composition for treating and / or preventing a central nervous system disease.

[0195] Preferably and in certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. Preferably and in certain embodiments, the recombinant transport peptide conjugate or the effector comprised by the transport peptide conjugate is a therapeutic agent for a central nervous system disease. Preferably and in certain embodiments, the transport peptide on the recombinant transport peptide conjugate or the transport peptide on the transport peptide conjugate binds to a transferrin receptor on a cell of a tissue barrier in the subject, and the binding of the transport peptide to the transferrin receptor induces transcytosis of the cell, thereby transporting the transport peptide and the therapeutic agent for the central nervous system disease across the tissue barrier. Preferably and in certain embodiments, the tissue barrier comprises a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier. Preferably and in certain embodiments, the subject is a mammal. More preferably and in certain preferred embodiments, the subject is a rodent or a human subject. Preferably and in certain embodiments, the modes of administration include intradermal, intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal, intrapulmonary, and transdermal delivery. Preferably and in certain embodiments, the central nervous system diseases include Alzheimer's disease, Parkinson's disease, cerebrovascular accident, vascular-related dementia, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, and spinal muscular atrophy.

[0196] Formulations suitable for administration in accordance with the present invention may include, among other things that may be known to those of skill in the art: aqueous and non-aqueous solutions, antioxidants, bacteriostatic agents, buffers, solutes affecting isotonicity, preservatives, solubilizing agents, stabilizers, suspending agents, thickening agents, or combinations thereof.

[0197] Additionally or alternatively, formulations suitable for administration in accordance with the present invention may include, as may be known to those of skill in the art: gels, PEG such as PEG 400, propylene glycol, saline, sachets;, water, other suitable liquids known in the art, or combinations thereof.

[0198] Additionally or alternatively, formulations suitable for administration in accordance with the present invention may include, as may be known to those of skill in the art: binders, buffers, calcium phosphate, cellulose, colloids such as colloidal silica, coloring agents, diluents, disintegrants, dyes, fillers, flavoring agents, gelatin, lactose, magnesium stearate, mannitol, microcrystalline gelatin, wetting agents, paraffin hydrocarbons, lozenges, polyethylene glycols, preservatives, sorbitol, starches such as corn starch, potato starch, or combinations thereof, stearic acid, sucrose, talc, triglycerides, or combinations thereof.

[0199] Additionally or in the alternative, formulations suitable for administration in accordance with the present invention may include, which may be known to those skilled in the art: alcohols such as benzyl alcohol or ethanol, benzalkonium chloride, buffers such as phosphate buffer, acetate buffer, citrate buffer, or combinations thereof, carboxymethylcellulose or microcrystalline cellulose, cholesterol, glucose, fruit juices such as grapefruit juice, milk, phospholipids such as lecithin, oils such as vegetable oil, fish oil, or mineral oil, or combinations thereof; other pharmaceutically compatible carriers known in the art; or combinations thereof.

[0200] Additionally or in the alternative, formulations suitable for administration in accordance with the present invention may include, which may be known to those skilled in the art: biodegradable, for example poly(lactic-co-glycolic acid) (PLGA) polymers, and degradation products of other entities that can be rapidly cleared from a biological system, or combinations thereof.

[0201] The formulations of the present invention may be administered in unit dose form, multi-dose form, or a combination thereof. They may be packaged in unit dose containers, multi-dose containers, or a combination thereof. The present invention may be present in ampoules, small capsules, capsules, granules, lozenges, powders, tablets, vials, emulsions, including but not limited to gum arabic emulsions, suspensions, or combinations thereof.

[0202] As used in the present invention, an "effective amount" or a "sufficient amount" of a substance is an amount sufficient to achieve a beneficial or desired result (including a clinical result), and thus, an "effective amount" depends on the circumstances in which it is applied. In the case of administering an immunogenic composition, the effective amount is an immunogenic effective amount, which includes an amount of the immunogenic composition of the present invention sufficient to elicit an immune response. In the case of administering a pharmaceutical composition, the effective amount is a pharmaceutically effective amount, which includes an amount of the pharmaceutical composition of the present invention sufficient to maintain or produce the desired physiological result. One or more doses of an effective amount may be administered.

[0203] As used in the present invention, the term "pharmaceutically effective amount" refers to an amount capable of or sufficient to maintain or produce a desired physiological result, including, but not limited to, treating, reducing, alleviating, eliminating, inhibiting, substantially preventing, or preventing, or combinations thereof, a disease, disorder, or combinations thereof. A pharmaceutically effective amount may include one or more doses administered sequentially or simultaneously. Those skilled in the art will know to adjust the dosage of the present invention to accommodate various types of formulations, including but not limited to, sustained release formulations. As used in the present invention, the term "prophylactic" refers to any aspect of a composition capable of substantially preventing or preventing a disease, disorder, or combinations thereof. As used in the present invention, the term "therapeutic" refers to any aspect of being able to treat, reduce, stop deterioration, slow down deterioration, beneficially alter, eliminate, or combinations thereof, a disease, disorder, or combinations thereof.

[0204] As used in the present invention, the term "dose" with respect to a composition refers to a measured portion of the composition taken (administered or received) by a subject at any given time.

[0205] As used in the present invention, the term "subject" refers to an animal, and more particularly to a non-human mammal and a human organism. Non-human animal subjects may also include prenatal forms of the animal, such as an embryo or fetus. Non-limiting examples of non-human animals include: horses, cows, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, pigs. In certain embodiments, the subject is a human. Human subjects may also include fetuses.

[0206] As used in the present invention, the term "subject" refers to any subject in need of treatment, particularly a mammalian subject, such as a human.

[0207] As used in the present invention, the terms "treat", "treating", or "treatment" include alleviating at least one of its symptoms, reducing its severity, or inhibiting its worsening. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. For a composition to be an effective treatment, it only needs to reduce the severity of a disease, disorder, or condition, reduce the severity of the symptoms associated therewith, or improve the quality of life of a patient or subject.

[0208] As used in the present invention, the terms "prevent", "preventing", or "prevention" refer to being able to substantially preclude, avoid, avert, prevent, stop, hinder, or a combination thereof the occurrence of any aspect of a disease, disorder, or a combination thereof, particularly by pre-emptive action.

[0209] In certain embodiments, the oligonucleotide therapeutic agent and / or composition of the present invention can be administered to a subject by a variety of routes of administration, including intradermal, intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal, pulmonary, and transdermal delivery, or topical administration to the eye, ear, skin, or mucosa. Alternatively, the antigen can optionally be administered ex vivo by direct exposure to cells, tissues, or organs derived from a subject (autologous) or another subject (allogeneic) in a biologically suitable liquid or solid carrier.

[0210] The meanings of the technical and scientific terms as described in the present invention can be clearly understood by those of ordinary skill in the art.

[0211] As used in the present invention, the terms "about", "approximately" or "substantially" when combined with a numerical value refer to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length in the range of 900 nm to 1100 nm.

[0212] As used in the present invention, the term "comprising" is open-ended, indicating that such embodiments may include additional elements. In contrast, the term "consisting of" is closed-ended, indicating that such embodiments do not include additional elements (except for trace impurities). The term "consisting essentially of" is partially closed-ended, indicating that such embodiments may also include elements that do not substantially change the basic characteristics of such embodiments.

[0213] When the applicant uses an open-ended conjunction such as "comprising" to define the invention or a part thereof, it should be readily understood that (unless otherwise specified), the specification should be interpreted as also using the conjunctions "consisting essentially of" or "consisting of" to describe the invention.

[0214] It should be noted that as used in the present invention and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, reference to "the polypeptide" includes one or more of the polypeptides recited and equivalents known to those skilled in the art, and the like. It should also be noted that the claims may be drafted to exclude any optional element. Thus, the present invention is intended as a basis for the prior use of exclusive terms such as "solely", "only" and the like when referring to claim limitations or using "negative" limitations.

[0215] In some cases, when using conventions such as "at least one of A, B, and C, etc.", generally such a construction is intended in the sense that those skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, and C" will include, but not be limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further understand that any disjunctive word and / or phrase that actually presents two or more alternative terms, whether in the description, claims or drawings, should be understood as contemplating the possibility of including one of the terms, either term, or both terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B".

[0216] The present invention is further illustrated by the following examples, which are provided for demonstration rather than limitation. Those skilled in the art should understand that, based on the disclosure of the present invention, various changes can be made to the disclosed embodiments without departing from the spirit and scope of the present invention, and similar or analogous results can still be obtained.

[0217] Example

[0218] The animal experiment protocols of the embodiments disclosed in the present invention have all been reviewed and approved by the Institutional Animal Care and Committee of the Development Center for Biotechnology (DCB) (Taipei, Taiwan, China) (Document No.: IACUC#2023-R403-006).

[0219] Example 1. Analysis of the binding ability between the transport peptide and the transferrin receptor

[0220] In this example, the affinity of the transport peptide described in 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 transport peptide were further substituted with different amino acids of the same nature to analyze the binding affinity of these mutant peptides for the transferrin receptor.

[0221] Materials and methods

[0222] The transport peptides (SEQ ID NO: 2 to SEQ ID NO: 30) of the present invention were respectively dissolved in PBS buffer at pH 7.4 at a concentration of 10 μg / ml. 80 μl of the solution was dropped onto a standard sensing chip (NanoAu-MM, InstantNanoBiosensors, Taiwan, China) pre-activated on the surface with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-Hydroxysuccinimide (NHS), so that the transport peptide was conjugated with the carboxyl group on the surface of the sensing chip. Transferrin receptor (TfR protein, ACROBiosystems, product number: CD1-H5243, USA) was loaded into the sensing 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. According to the change in light intensity, the interaction parameter KD between molecules was calculated using a FOPPR system detector (FOPPR system D200(Id M3), Taiwan, China).

[0223] Results

[0224] The affinity between the transport peptide and transferrin receptor of the present invention is expressed by the KD value (Kinetic data). The higher the KD value, the weaker the binding between the two molecules; on the contrary, the lower the KD value, the stronger the binding between the two molecules (KD value < 300 nM indicates that the tested peptide has a strong binding affinity for TfR). As shown in Table 2, each of the transport peptides (SEQ ID NO: 2 to SEQ ID NO: 30) disclosed in the present invention has a strong binding affinity for transferrin receptor. The results confirm that each site of the transport peptide (SEQ ID NO: 1) disclosed in the present invention can be replaced by several specific amino acids without losing its binding affinity for TfR.

[0225] Table 2. Binding affinity of transport peptide for transferrin receptor

[0226]

[0227]

[0228] Example 2. In vitro transcytosis analysis of transport peptide

[0229] In this example, the transport efficiency of the transport peptide of the present invention on Caco-2 monolayer cells was tested to analyze the penetration effect of the transport peptide of the present invention on tissue barriers and to analyze the ability of the transport peptide to induce transcytosis in the cells.

[0230] Materials and Methods

[0231] Cell Culture

[0232] Caco-2 cells were cultured at a density of 2x104 cells / cm2 in a 24-well plate (24-transwell inserts, BDFalcon, USA, product number: 353495) with Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were cultured for 21 days with DMEM containing 10% fetal bovine serum to form a monolayer. The transepithelial electrical resistance (TEER) was measured and when it reached at least 500 - 600 Ω·cm2, it was considered a qualified monolayer for subsequent cell penetration experiments.

[0233] High Performance Liquid Chromatography (HPLC) Analysis

[0234] HPLC analysis was performed using an HPLC analyzer system (Water ACQUITY Arc system). The column temperature of the C-18 column (Waters XSelect HSS T3 column) was maintained at 40°C. The mobile phase consisted of (A) an aqueous solution of 0.1% trifluoroacetic acid and (B) 100% methanol. The separation process was carried out according to a gradient program: 0 minutes - 10% (B), 6 minutes - 30% (B), 12 minutes - 50% (B), 18 minutes - 90% (B), 23 minutes - 10% (B). The flow rate of the mobile phase was 1 mL / min, the injection volume of the sample was 20 μL, and the total running time was completed within 30 minutes.

[0235] In Vitro Transcytosis Analysis

[0236] Using a TEER value of 680 Ω cm 2The Caco-2 monolayer culture medium was equilibrated with serum-free DMEM medium. At 37 °C, the transport peptide (PT-034, SEQ ID NO: 19) of the present invention at a concentration of 10 μM was placed on the upper layer of the Caco-2 monolayer culture medium, and the solutions of the upper layer (Upper) and the lower layer (Lower) of the Caco-2 monolayer were sampled at 0, 0.25, 0.5, 1, 2, and 6 hours respectively. The peptides in the sample solutions were purified by solid-phase extraction (SPE) using C-18 tips (Pierce, product number: 87784, USA), and each sample was supplemented with PT-034 at a final concentration of 11.43 μM as an internal control. 300 μL of the sample was adsorbed onto the C18 tips and eluted with 100 μL of methanol, and finally the samples were analyzed by HPLC. The content of the transport peptide in the sample solution was calculated by calculating the HPLC peak area at a retention time of 16.9 minutes, and the content of the transport peptide in each sample was calculated by establishing a standard curve. The concentrations of the transport peptide in the upper layer and the lower layer were calculated to obtain the proportion of the transport peptide penetrating the Caco-2 cell monolayer.

[0237] Results

[0238] As Figure 1 shown, at 0.25, 1, 2, and 6 hours, approximately 8%, 40%, 42%, and 56% of the transport peptide (SEQ ID NO: 19) penetrated the Caco-2 cell monolayer respectively. The results show that the transport peptide of the present invention has the ability to transport through tissue barriers and can effectively induce transcytosis in the cells.

[0239] Example 3. Analysis of the in vivo imaging system of the transport peptide

[0240] In this example, the transport peptide of the present invention was administered to mice, and the distribution of the transport peptide in the mice was detected by an in vivo fluorescence imaging system (IVIS).

[0241] Materials and methods

[0242] Conjugation of the peptide and the fluorescent dye

[0243] 1 mg of the transport 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) were separately dissolved in 1 mL of a 50 mM sodium bicarbonate / bicarbonate buffer to a final concentration of 1 mg / mL. 50 μL of a 10 mg / mL fluorescent dye (VivoTag 680XL, PerkinElmer, USA) was added to the 1 mL of the transport peptide solution, and the reaction was carried out in the dark at room temperature for 1 hour. The product conjugated with the dye was filtered using a 3 KDa centrifugal filter device (Amicon Ultra-0.5 Centrifugal filter devices, Millipore, USA) and centrifuged at 14,000 xg for 15 minutes. The transport peptide conjugated with the fluorescent dye was diluted 1-fold with PBS to a final volume of 0.2 mL.

[0244] Animal experiments

[0245] BALB / c mice at 7 - 8 weeks of age (Lexicon Biotech Inc., Taiwan, China) were divided into a control group (n = 1) and an experimental group (n = 3). The control group mice were administered 50 μL of the fluorescent dye by tail vein injection. The experimental group mice were administered 50 μL of the above-mentioned transport peptide conjugated with the fluorescent dye by tail vein injection. After injection, the mice were photographed using an optical imaging device (U-OI, MlLabs, Netherlands) at 0.5, 1, 2, 4, 6, and 24 hours for IVIS imaging analysis, with an excitation wavelength of 631 nm, an emission wavelength of 710 nm, and an exposure time of 10 seconds. Further quantification of the fluorescence signal in the mouse brain was carried out.

[0246] Results

[0247] 3.1 In vivo biodistribution of PT-034

[0248] As Figure 2A shown, at 1, 2, 4, and 6 hours after injection, the fluorescence signal was enriched in the brains and spines of the experimental group mice administered with 10 mg / kg of the transport peptide PT-034 (SEQ ID NO:19) conjugated with the fluorescent dye; in contrast, such a fluorescence signal distribution was not present in the control group (Dye) mice. Further quantification of the fluorescence signal in the mouse brain was carried out. As Figure 2B shown, compared with the control group, a high amount of fluorescence signal could be observed in the brains of the experimental group mice at 1 hour after injection, and the half-life of the fluorescence in the mouse brain was approximately 5 hours. The experimental results confirmed that the transport peptide of the present invention has the efficacy of targeting the brain and spine and can be used as an effective drug delivery system.

[0249] 3.2 In vivo biodistribution of PT-001, PT-025 and PT-031

[0250] As Figure 3A shown, at 0.5, 1, 2, 4, and 6 hours after injection, the fluorescence signals were enriched in the brains and spines of the experimental group mice administered with 1 mg / kg of the transport peptides PT-001 (SEQ ID NO:2), PT-025 (SEQ ID NO:11), and PT-031 (SEQ ID NO:17) conjugated with fluorescent dyes, respectively. Further quantification of the fluorescence signals in the mouse brains was performed. As Figure 3B shown, a high amount of fluorescence signals could be observed in the brains of the experimental group mice at 0.5 hour after injection, and the half-life of the fluorescence in the mouse brains was approximately 5 hours. The experimental results confirmed that the peptides (SEQ ID NO:2 to SEQ ID NO:30) obtained by substituting different amino acids at different amino acid sites of the transport peptide (SEQ ID NO:1) of the present invention had the efficacy of targeting the brain and spine and could be used as an effective drug delivery system.

[0251] Example 4. Analysis of the brain transport efficiency of the transport peptide

[0252] In this example, by detecting the transport efficiency of the transport peptide of the present invention in the brain, it was confirmed that the transport peptide of the present invention had the efficacy of transporting through the blood-brain barrier.

[0253] Materials and methods

[0254] Mice (n = 2) were intravenously injected with 10 mg / kg of the transport peptide (PT-034, SEQ ID NO:19) of the present invention. One hour after injection, the brain tissues were collected and ground in 1.3 mL of 50 mM sodium bicarbonate / bicarbonate buffer. The brain tissue homogenate was centrifuged at 3000 rpm for 5 minutes, and the supernatant was taken and then centrifuged at 14000 rpm for 15 minutes. 0.4 mL of ice-cold methanol was added to 0.1 mL of the supernatant of the brain tissue homogenate, and it was shaken for 10 minutes and then centrifuged at 14000 rpm for 15 minutes. Subsequently, the brain tissue supernatant was subjected to HPLC analysis, and the HPLC analysis method was as described in Example 2. The HPLC peak area at a retention time of 16.9 minutes was calculated, and the content of the transport peptide in each sample was calculated by establishing a standard curve.

[0255] Results

[0256] As shown in Table 3, in the whole brain tissues (Brain Tissue-1 and Brain Tissue-2) of mice in each experimental group, the contents of the transport peptide (PT-034) were 25.5 μg and 25.9 μg, respectively. When the contents were converted to the percentage of injection dose per gram of brain tissue (%ID / g of brain, injection dose / brain tissue weight), they were 22.09% and 23.48%, respectively, indicating that the average transport efficiency of the transport peptide to the brain was approximately 22.79%ID / g. The experimental results confirmed that the transport peptide of the present invention has the efficacy of transporting through the blood-brain barrier and reaching the brain.

[0257] Table 3. Intracerebral transport efficiency table of transport peptide

[0258]

[0259] Example 5. In vivo imaging system analysis of transport peptide conjugate (conjugated with antibody)

[0260] In this example, the transport peptide of the present invention was conjugated with an antibody to form a transport peptide conjugate. Then, the transport peptide conjugate was administered to mice, and the distribution of the transport peptide conjugate in the mice was detected by an in vivo imaging system (IVIS). In this example, the transport peptide conjugate was administered by intravenous injection and oral administration, respectively, to explore the efficacy of different administration routes.

[0261] Materials and methods

[0262] Preparation of transport peptide-conjugated antibody labeled with fluorescent dye

[0263] Adjust the concentration of the anti-Her-2 antibody (trastuzumab, Selleckchem, USA) to 1 mg / mL with 100 mM carbonate buffer (carbonate / bicarbonate buffer). Dissolve the crosslinker (bis(sulfosuccinimidyl)suberate, Thermo Scientific, USA) in water and adjust the concentration to 25 mM. Mix 1 mL of the above anti-Her-2 antibody, 11 μL of the crosslinker solution, and 8.4 μL of the transport peptide described in the present invention (concentration 1 mg / mL), and react at room temperature for 30 minutes to carry out a covalent synthesis reaction. Centrifuge the reaction mixture through a 3 kDa filter at 14,000 xg for 15 minutes to obtain the transport peptide conjugate (conjugated with the anti-Her-2 antibody). Adjust the volume of the product to 1 mL with 50 mM carbonate buffer, add 10 μL of a fluorescent dye (VivoTag680XL) with a concentration of 10 mg / mL, let it stand in the dark at room temperature for 1 hour, then centrifuge through a 3 kDa filter at 14,000 xg for 15 minutes, and finally adjust the total volume to 0.2 mL with 100 mM carbonate buffer. This product is the transport peptide conjugate labeled with a fluorescent dye (Ab-PT-D).

[0264] Preparation of the antibody labeled with a fluorescent dye

[0265] Add 10 μL of a fluorescent dye (VivoTag 680XL) with a concentration of 10 mg / mL to the anti-Her-2 antibody solution, let it stand in the dark at room temperature for 1 hour, then centrifuge through a 3 kDa filter at 14,000 xg for 15 minutes to label the antibody with the fluorescent dye (Ab-D). Adjust the final volume to 0.2 mL with 50 mM carbonate buffer for the negative control group of this example.

[0266] Animal experiments

[0267] Administer the above fluorescent dye-labeled transport peptide conjugate to 7-8-week-old experimental mice (BALB / c mice, Lesco Biotechnology Co., Ltd., Taiwan, China). After 0.5, 1, 2, 4, 6, and 24 hours, use an optical imaging device (U-OI, MlLabs, Netherlands) to take pictures for IVIS imaging analysis, and analyze with an excitation wavelength of 631 nm, an emission wavelength of 710 nm, and an exposure time of 10 seconds. Further quantify the fluorescence signal in the brain.

[0268] Results

[0269] 5.1 Intravenous injection

[0270] The transit peptide used in this experiment was PT-034 (SEQ ID NO:19). Mice in the control group (Ab-D) (n = 3) were administered the above-mentioned antibody labeled with fluorescent dye via tail vein injection at a dose concentration of 10 mg / kg, and mice in the experimental group (Ab-PT-D) (n = 3) were administered the above-mentioned transit peptide conjugate labeled with fluorescent dye via tail vein injection at a dose concentration of 10 mg / kg. As Figure 4A shown, the imaging results showed that after the experimental group (Ab-PT-D) was administered the transit peptide conjugate labeled with fluorescent dye, the fluorescent signal was enriched in the brain and spine, and had a stronger fluorescent signal distribution compared to the control group. As Figure 4B shown, in this example, the fluorescent signal in the mouse brain was further quantified. Compared with the control group, a higher fluorescent signal was observed in the experimental group in the brain. This experiment confirmed that after conjugating the transit peptide of the present invention with an antibody, the formed transit peptide conjugate still retained the efficacy of targeting the brain and spine and could be used as an effective drug delivery system.

[0271] 5.2 Oral administration

[0272] The transit peptide used in this experiment was PT-034 (SEQ ID NO:19). Mice in the intravenous injection group (n = 2) were administered the above-mentioned transit peptide conjugate labeled with fluorescent dye via tail vein injection at a dose concentration of 0.83 mg / kg, and mice in the oral administration group (n = 2) were administered the above-mentioned transit peptide conjugate labeled with fluorescent dye via oral administration at a dose concentration of 0.83 mg / kg. Please refer to Figure 5A . The imaging results showed that after the intravenous injection group and the oral administration group were administered the transit peptide conjugate labeled with fluorescent dye, the fluorescent signal was shown in the brain or spine. Please refer to Figure 5B . In the mice administered via the oral route (oral administration group), obvious fluorescent signals could be detected in the brain, indicating that the transit peptide of the present invention could be administered via the oral route. Please refer to Figure 5C . In this example, the fluorescent signal in the mouse brain was further quantified. The results showed that fluorescent signals could be observed in the brain after both intravenous injection and oral administration, confirming that the transit peptide of the present invention could be administered via the oral route or by intravenous injection, and both had the efficacy of targeting the brain and could be used as an effective drug delivery system.

[0273] The above results confirmed that the transit peptide conjugate of the present invention could maintain its efficacy whether administered via intravenous injection or oral administration. When the transit peptide of the present invention was administered orally, it would bind to the transferrin receptor on the cells of the gastrointestinal mucosal barrier, induce cell transcytosis to occur, and be transported through the gastrointestinal mucosal barrier into the body and then target the brain. The transit peptide could also be transported through the blood-brain barrier by the same mechanism.

[0274] In Vivo Imaging System Analysis of a Translocation Peptide Conjugate (Conjugated with Lipid Nanoparticles)

[0275] In this example, the translocation peptide of the present invention was conjugated with lipid nanoparticles (LNP) to form a translocation peptide conjugate, and then the translocation peptide conjugate was administered to mice, and the in vivo distribution of the translocation peptide conjugate was detected by an in vivo imaging system (IVIS).

[0276] Materials and Methods

[0277] Preparation of Lipid Nanoparticles Labeled with a Fluorescent Dye

[0278] 2000 μg of lipid nanoparticles (LNP-102, ABP Biosciences, USA) dissolved in an ethanol solution and 200 μg of a fluorescent dye (VivoTag 680XL) dissolved in a 50 mM sodium acetate solution were mixed, gently stirred at room temperature for 30 minutes, and then dialyzed against PBS for 24 hours in a dialysis cassette with a 2K molecular weight cut-off (Slide-A-Lyzer, ThermoFisher Scientific, USA) to prepare lipid nanoparticles labeled with a fluorescent dye (LNP-D).

[0279] Preparation of Translocation Peptide-Conjugated Lipid Nanoparticles Labeled with a Fluorescent Dye

[0280] 0.1 mL of the translocation peptide of the present invention at a concentration of 1 mg / mL was mixed with 5.8 μL of a cross-linker solution (EZ-Link TFP Ester-PEG4-DBCO, ThermoFisher Scientific, USA) (concentration: 20 mg / mL) and reacted at room temperature for 1 hour. A total of 106 μL of the above solution was added to 475 μL of the above lipid nanoparticle solution labeled with a fluorescent dye and reacted at room temperature for 1 hour to prepare translocation peptide-conjugated lipid nanoparticles labeled with a fluorescent dye (LNP-PT-D).

[0281] Animal Experiments

[0282] Experimental mice (BALB / c mice) at 7-8 weeks of age were divided into a control group and an experimental group. Mice in the control group (n = 3) were intravenously injected via the tail vein with the above-mentioned lipid nanoparticles labeled with fluorescent dye (LNP-D) at a dose concentration of 10 mg / kg, and mice in the experimental group (n = 3) were intravenously injected via the tail vein with the above-mentioned transport peptide conjugate labeled with fluorescent dye (LNP-PT-D) at a dose concentration of 10 mg / kg. After 0.5, 1, 2, 4, 6, and 24 hours, an optical imaging device (U-OI) was used for shooting to perform IVIS imaging analysis, and the analysis was carried out at an excitation wavelength of 631 nm, an emission wavelength of 710 nm, and an exposure time of 10 seconds. Furthermore, the fluorescence signal in the mouse brain was quantified.

[0283] Results

[0284] The transport peptide used in this experiment was PT-034 (SEQ ID NO:19), as Figure 6A shown. The imaging results showed that after the administration of the transport peptide conjugate labeled with fluorescent dye in the experimental group (LNP-PT-D), the fluorescence signal was enriched in the brain and spine, and compared with the control group (LNP-D), the experimental group had a stronger fluorescence signal distribution. As Figure 6B shown, a higher fluorescence signal was observed in the brain of the experimental group (LNP-PT-D) compared with the control group (LNP-D). This experiment confirmed that after conjugating the transport peptide of the present invention with a lipid nanoparticle, the formed transport peptide conjugate still retained the efficacy of targeting the brain and spine and could be used as an effective drug delivery system.

[0285] In summary, the transport peptide of the present invention was experimentally confirmed to have a binding affinity with the transferrin receptor. After the transport peptide of the present invention binds to the transferrin receptor on the cells located in the tissue barrier, it will induce the transcytosis of the cells, thereby transporting the transport peptide and its conjugate through the tissue barrier. The transport peptide of the present invention (SEQ ID NO:1) was experimentally confirmed that even after some amino acid positions were substituted with amino acids of the same nature, it still retained the binding affinity with the transferrin receptor. The transport peptide of the present invention can be conjugated with an effector to form a transport peptide conjugate. It was experimentally confirmed that the transport peptide conjugate of the present invention retained the ability to bind to the transferrin receptor, and the transport peptide conjugate of the present invention had the efficacy of transporting the effector through the tissue barrier, effectively overcoming the problem that drugs are difficult to pass through the blood-brain barrier, and has broad application value for the treatment and / or prevention of central nervous system diseases, and also contributes to the development of related clinical medical fields.

[0286] Of course, many changes and modifications can be made to the above embodiments of the present invention without departing from the scope of the present invention. Therefore, for the purpose of promoting progress in the scientific and useful arts, the present invention is disclosed and is intended to be limited only by the scope defined by the appended claims.

Claims

1. A transit peptide, characterized in that, Composed of the amino acid sequence shown in SEQ ID NO:1, and the transport peptide can bind to a transferrin receptor.

2. The transit peptide according to claim 1, characterized in that, The amino acid sequence of the transport peptide is selected from the group consisting of the amino acid sequences shown in SEQ ID NO:2 to SEQ ID NO:30 and variant sequences having at least 70% homology with any of the amino acid sequences shown in SEQ ID NO:2 to SEQ ID NO:30 and capable of binding to the transferrin receptor.

3. A nucleic acid encoding the transport peptide according to claim 1.

4. The nucleic acid according to claim 3, wherein, The nucleic acid is deoxyribonucleic acid or ribonucleic acid.

5. A carrier, characterized in that, Comprising the nucleic acid according to claim 3.

6. The carrier according to claim 5, characterized in that The vector is an adeno-associated virus vector.

7. The carrier according to claim 5, wherein Further comprising a nucleic acid encoding an effector.

8. The carrier according to claim 7, wherein The effector is selected from at least one of the group consisting of peptides, proteins, antibodies, virus particles, liposomes, endosomes, exosomes, ligands, eukaryotic cells, prokaryotic cells, and microspheres.

9. A recombinant transit peptide conjugate, characterized in that, Expressed by the vector according to claim 7.

10. A recombinant host cell, characterized in that, Comprising a component selected from the group consisting of: The peptide according to claim 1, the nucleic acid according to claim 3, and the vector according to claim 5.

11. A transit peptide conjugate, characterized in that, Composed of the transport peptide according to claim 1 and an effector, and the transport peptide and the effector are conjugated in a covalent or non-covalent manner.

12. The transit peptide conjugate according to claim 11, wherein, The effector is selected from at least one of the group consisting of siRNA, shRNA, microRNA, double-stranded RNA, single-stranded RNA, DNA, oligonucleotides, aptamers, genes, peptides, proteins, antibodies, small chemical molecules, large chemical molecules, virus particles, liposomes, endosomes, exosomes, nanoparticles, lipid nanoparticles, dendrimers, ligands, eukaryotic cells, prokaryotic cells, microspheres, nanogels, and biocompatible nanocapsules.

13. A composition, characterized in that, Comprising: A transport peptide according to claim 1; A recombinant transport peptide conjugate according to claim 9; or A transport peptide conjugate according to claim 11.

14. The composition according to claim 13, wherein, Further comprising a pharmaceutically acceptable carrier.

15. Use of a transit peptide as claimed in claim 1 in the preparation of a pharmaceutical composition for transporting an effector to a target, characterized in that, The target is in vivo or in vitro.

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

17. The use according to claim 15, wherein, The transport peptide and the effector form a recombinant transport peptide conjugate according to claim 9 by expressing a nucleic acid encoding the transport peptide and the effector.

18. The use according to claim 15, wherein The transport peptide and the effector must reach the target through a tissue barrier, and the transport peptide binds to a transferrin receptor on a cell of the tissue barrier.

19. The use according to claim 18, characterized in that, The binding of the transport peptide to the transferrin receptor induces transcytosis of the cell, and transports the transport peptide and the effector through the tissue barrier to reach the target.

20. The use according to claim 18, wherein The tissue barrier includes a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier.

21. The use according to claim 15, characterized in that, The target is a cell expressing a transferrin receptor, and the transport peptide transports the effector to the target by binding to the transferrin receptor expressed on the target cell.

22. Use of a transport peptide as claimed in claim 1 in the preparation of a pharmaceutical composition for transporting an effector through a tissue barrier of a subject.

23. The use according to claim 22, characterized in that, The transport peptide is conjugated to the effector covalently or non-covalently to form a transport peptide conjugate as claimed in claim 11.

24. The use according to claim 22, wherein, The transport peptide and the effector are formed into a recombinant transport peptide conjugate as claimed in claim 9 by expressing a nucleic acid encoding the transport peptide and the effector.

25. The use according to claim 22, characterized in that, The tissue barrier includes a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier.

26. The use according to claim 22, characterized in that, A transferrin receptor is present on a cell of the tissue barrier.

27. The use according to claim 26, wherein, The transport peptide binds to the transferrin receptor, and the binding of the transport peptide to the transferrin receptor induces transcytosis of the cell, thereby transporting the transport peptide and the effector through the tissue barrier.

28. Use of a transport peptide as claimed in claim 1 in the preparation of a pharmaceutical composition for treating and / or preventing central nervous system diseases.

29. The use according to claim 28, wherein, The transport peptide is conjugated to an effector covalently or non-covalently to form a transport peptide conjugate as claimed in claim 11.

30. The use according to claim 28, characterized in that, The transport peptide and the effector are formed into a recombinant transport peptide conjugate as claimed in claim 9 by expressing a nucleic acid encoding the transport peptide and the effector.

31. The use according to claim 29 or 30, characterized in that, The effector is a therapeutic agent for central nervous system diseases.

32. The use according to claim 29 or 30, characterized in that, The transport peptide conjugate or the recombinant transport peptide conjugate is administered to a subject in need thereof.

33. The use according to claim 28, wherein, The transport peptide binds to a transferrin receptor on a cell of a tissue barrier in the subject's body, and the binding of the transport peptide to the transferrin receptor induces transcytosis of the cell, thereby transporting the transport peptide and the therapeutic agent for central nervous system diseases through the tissue barrier.

34. The use according to claim 33, wherein, The tissue barrier includes a blood-brain barrier, a mucosal barrier, and a gastrointestinal barrier.

35. The use according to claim 28, characterized in that, The central nervous system diseases include Alzheimer's disease, Parkinson's disease, cerebrovascular accident, vascular-related dementia, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's chorea, and spinal muscular atrophy.

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