Molecular transport system to central nervous system

Abstract

To provide a selection platform that exploits the physiology of the choroid plexus to identify molecular transport system (MTS) peptides capable of holding various functional cargos in the cerebrospinal fluid and distributing the cargos to cells of the CNS.SOLUTION: Provided are MTS peptides or targeting peptides comprising a specific amino acid sequence. Provided are compositions comprising a peptide, the peptide comprising a first MTS peptide conjugated to a cargo. Methods of using the MTS peptides are also provided. For example, provided is a method for transporting cargo to the CNS, the method comprising administering one or more of the disclosed compositions to a subject in need thereof, the peptide conjugated to the cargo entering the CNS.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 991,465, filed March 18, 2020. No. 6,299,499, filed on Oct. 1, 2004, which is hereby incorporated by reference in its entirety.

[0002] Reference to sequence listing "37794_" created on March 16, 2021, with a size of 3,389 bytes A text file named "0096P1_Sequence_Listing.txt" The sequence listing filed on March 18, 2021, is in compliance with 37 CFR 1.52(e)( 5) is incorporated herein by reference. [Background technology]

[0003] Access to the central nervous system (CNS) is a bottleneck in the development of neurotherapeutics. This leads to a series of specialized and highly selective cellular interactions that protect the central nervous system (CNS). This is due to the blood-brain barrier (BBB), which is a barrier to normal physiology. prevents the entry of many chemical entities, including neurotherapeutics, into the brain. Less than 5% of infections cross the BBB. Furthermore, newer biological therapies such as antibody and gene therapy Law is essentially excluded from the CNS due to the BBB. Despite these findings, no general solution for delivery to the CNS has been created. As a result, many CNS disorders have no treatment options.

[0004] Currently, there are several approaches used to deliver drugs to the CNS. One approach is to use BBB-permeable compounds. The problem with using BBB-permeable compounds is that they have poor biodistribution properties and off-target effects. (as they often tend to be highly lipid soluble) Also, fewer than 5% of small molecules penetrate the BBB, so most BBB-permeable compounds There are no BBB-permeable drugs. The indications for BBB-permeable drugs are also limited.

[0005] Another approach to access the CNS is by direct injection into the spinal cord or brain. This method is invasive and carries the risk of structural damage to the surrounding tissue and infection. The risk of

[0006] Disruption of the BBB is another method used. This method allows the delivery of compounds, cells, and This method allows for the mass transport of pathogens and neuronal dysfunction in addition to structural damage. may cause harm.

[0007] Intranasal delivery has also been used. This method is limited to lipophilic small molecule drugs. Intranasal delivery has been shown to have poor distribution through the CNS. Absorption varies between patients.

[0008] Receptor-mediated delivery (Trojan horse) methods have also been used. The receptor is not universal because it is only effective for one cargo. They can be expressed intracellularly, have poor CNS uptake, and are toxic. Receptor-mediated delivery of α-amyloids does not have cell specificity when delivered across the BBB and is not readily accessible to the CNS. The overall distribution is inconsistent.

[0009] Blood-cerebrospinal fluid (BCS) delivery provides unique development opportunities to influence drug and macromolecule transport into the CNS. There are no known choroid plexus transport agents or alternative methods for transporting cargo across the cerebrospinal fluid barrier. Accordingly, compositions and methods for targeting the CNS are disclosed herein. Summary of the Invention

[0010] Carries a variety of functional cargoes in the cerebrospinal fluid, which can distribute the cargo to cells of the CNS Harnessing choroid plexus physiology to identify molecular transport system (MTS) peptides capable of Described herein are selection platforms for delivering cargo (e.g., biological Disclosed herein are MTS peptides for CNS delivery of therapeutically active cargoes.

[0011] MTS peptides or targeting peptides are disclosed herein. Disclosed are MTS peptides comprising any one of the amino acid sequences.

[0012] Also disclosed are peptides comprising an MTS peptide conjugated to a cargo.

[0013] A composition comprising a peptide, wherein the peptide is a first peptide conjugated to a cargo. Compositions comprising MTS peptides are disclosed. The peptide comprises a first MTS peptide conjugated to a cargo, The peptide comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 12. will be done.

[0014] A method for transporting cargo to the CNS, comprising administering one or more of the disclosed compositions to to a subject in need thereof, wherein the peptide conjugated to a cargo comprises: In some embodiments, a method is disclosed for conjugating a cargo to the CNS. The induced peptides enter the choroid plexus.

[0015] A method of treating a CNS disorder or injury, comprising administering to a subject a therapeutically effective amount of any of the disclosed peptides or compositions. and administering one or more of the cargo to a subject in need thereof, wherein the cargo is a CNS disorder or injury. A method is disclosed, which is a wound treatment.

[0016] A method of imaging the CNS, comprising administering one or more of the disclosed peptides or compositions to a subject to a subject in need thereof, wherein the cargo is an imaging agent. do.

[0017] Additional advantages of the disclosed methods and compositions are described in part in the following description and are set forth from the description. It may be understood, in part, or learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions reside in the elements and combinations particularly pointed out in the appended claims. This will be realized and achieved by the combination of the general description given above and the detailed description given below. Both disclosures are exemplary and explanatory only and are not intended to limit the invention as claimed. Please understand that this is not the case.

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate the disclosed methods and Several embodiments of the compositions are illustrated and, together with the description, provide principles of the disclosed methods and compositions. It serves to explain. [Brief explanation of the drawings]

[0019] [Figure 1]Schematic diagram of the brain with the vascular endothelial barrier (commonly referred to as the blood-brain barrier (BBB)) and choroid plexus. CSF = cerebrospinal fluid, CCSFB = choroidal cerebrospinal fluid barrier. [Figure 2] Schematic diagram showing key features of the choroid plexus as a transport point into the CNS. The structural features of fenestrated capillaries and the transcytotic activity of choroid plexus epithelium are responsible for the structural differences between the choroid plexus and the BBB. [Figure 3] 1 shows an experimental design for unbiased selection to identify MTSs that mediate functional transport activity. [Figure 4] 1 shows the experimental design for validation of in vivo delivery of MTS to the CNS. [Figure 5] This shows how the MTS system was developed. [Figure 6] 1 shows tissue staining of the CPEC4 phage clone in the choroid plexus and ventricle, which is a phage clone that expresses the CPEC4 peptide (SEQ ID NO: 2). [Figure 7] 1 shows tissue staining of CPEC4 phage clones outside the ventricular system in the brain. [Figure 8] Peptide engineering is shown to improve solubility and stability while maintaining transport capacity. [Figure 9] We demonstrate that the Transwell model using primary human choroid plexus epithelial cells maintains the characteristics of the CNS barrier. [Figure 10] An unbiased selection is presented to identify MTSs that mediate functional transport activity. [Figure 11] Transwell selection of human primary choroid plexus epithelial cells. [Figure 12] 1 shows validation of CPEC4 phage clone transport through human choroid plexus cells in an in vitro system. [Figure 13] The dimeric CPEC4 peptide is shown. [Figure 14] 1 shows validation of CPEC4 phage clone transport through human choroid plexus cells in an in vitro system. [Figure 15]This shows that the CPEC4 phage clone and MTS_CPEC4_V1 are transported through the rat choroid plexus in vitro. The CPEC4 phage clone is a phage clone that displays the CPEC4 peptide (SEQ ID NO: 2). MTS_CPEC4_V1 is a synthetic peptide of SEQ ID NO: 2. [Figure 16] Validation of the in vivo delivery system in the CNS is shown. [Figure 17] Figure 1 shows that CPEC4 phage clones preferentially accumulate in the CSF of rats compared to non-targeted empty phage clones. [Figure 18] 1 shows that MTS-CPEC4_V2 accumulates in the CSF of rats. MTS_CPEC4_V2 contains an MTS peptide having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO: 1). [Figure 19] The difference between MTS_CPEC4 before and after stabilization is shown. MTS_CPEC4 contains an MTS peptide with the sequence DGYKLQTSLDWQMWNP (SEQ ID NO: 2). [Figure 20]

[0039] Figure 1 shows the synthesis of MTS_CPEC4_V2 using an isoacyl Thr-Ser dipeptide. MTS_CPEC4_V2 contains an MTS peptide having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO: 1). [Figure 21] The transport rate using the MTS_CPEC4_V2 dimer is shown. The MTS_CPEC4_V1 dimer contains two MTS peptides, each with the sequence DGYKLQTSLDWQMWNP (SEQ ID NO: 2). The MTS_CPEC4_V2 monomer contains an MTS with the sequence DAYKLQTSLDWQMWNP (SEQ ID NO: 1). The MTS_CPEC4_V2 dimer contains two MTS peptides, each with the sequence DAYKLQTSLDWQMWNP (SEQ ID NO: 1). [Figure 22] This shows that MTS_CPEC4_V2 is stable in serum. MTS_CPEC4_V2 contains an MTS peptide having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO: 1). [Figure 23]1 shows the in vivo use of MTS_CPEC4_V2. MTS_CPEC4_V2 contains an MTS peptide having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO: 1). [Figure 24] Experimental design for in vitro and in vivo selection of lead MTS peptides. [Figure 25] We demonstrate that the MTS_Z310-2 and MTS_Z310-5 peptides transport protein cargo across rat choroid plexus epithelial cells. MTS_Z310-2 contains an MTS peptide with the sequence FPSWTSKNQQWTNQRQ (SEQ ID NO: 4). MTS_Z310-5 contains an MTS peptide with the sequence SKETYSMNAQRQHERS (SEQ ID NO: 7). [Figure 26] The MTS_Z310-5 dimeric peptide is shown to enter the CSF and be observed in the ventricular system of the brain. The MTS_Z310-5 dimeric peptide contains two MTS peptides, each with the sequence SKETYSMNAQRQHERS (SEQ ID NO: 7). [Figure 27] We demonstrate that the MTS_Z310-2 dimeric peptide enters the CSF and is observed in the ventricular system of the brain. MTS_Z310-2 contains two MTS peptides, each with the sequence FPSWTSKNQQWTNQRQ (SEQ ID NO: 4). [Figure 28] 1 shows how dimeric MTSs can be conjugated together or to cargo. [Figure 29] Examples of MTS chimeric molecules are shown. DETAILED DESCRIPTION OF THE INVENTION

[0020] The disclosed methods and compositions are described in the following detailed description of specific embodiments and the methods and compositions included therein. The present invention will be more readily understood by reference to the following examples and the accompanying drawings and accompanying description. It can be done.

[0021] The disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques, or the like, unless otherwise specified. It is understood that the present invention is not limited to the techniques or particular reagents, and as such may vary. Also, the terminology used herein is for the purpose of describing particular embodiments only. However, it should be understood that this is not intended to be limiting.

[0022] Can be used in conjunction with the disclosed methods and compositions Materials, compositions that can be used in the preparation of, or are products of, These and other materials are disclosed herein, and combinations of these materials are When disclosing combinations, subsets, interactions, groups, etc., various individual and A specific reference to each combination and permutation of the set may not be explicitly disclosed. However, it is to be understood that each is specifically contemplated and described herein. For example, a peptide is disclosed and discussed, and several molecules containing peptides are When several possible modifications are discussed, the peptide and each and every possible modification are Combinations and permutations of are specifically contemplated unless specifically indicated to the contrary. Thus, one of molecules A, B, and C, and one of molecules D, E, and F, and combinations thereof. When an example molecule is disclosed and an AD is disclosed, each is individually listed. Each of these is contemplated individually and collectively. Each of E, AF, BD, BE, BF, CD, CE, and CF is specifically It is contemplated that from the disclosure of A, B, and C; D, E, and F, and combination example AD, Likewise, any subset or combination of these are also specifically contemplated and disclosed. Thus, for example, AE, BF, and C- Subgroups of E are specifically contemplated, including A, B, and C; D, E, and F, and combinations thereof. It should be considered as disclosed from the disclosure of the combined example AD. The disclosed compositions include, but are not limited to, steps in methods of making and using the disclosed compositions. , applies to all aspects of this application. Accordingly, various additional steps that may be performed are If present, each of these additional steps is not essential to any particular implementation of the disclosed methods. It may be practiced with any embodiment or combination of embodiments, and the It is understood that each should be considered specifically contemplated and disclosed. sea ​​bream.

[0023] A.Definition The disclosed methods and compositions are based on the specific methodology, protocols, and experiments described therein. It should be understood that the term "medicinal agent" is not limited to drugs and can vary. The terms used herein are for the purpose of describing particular embodiments only and are defined by the appended claims. It is also understood that the present invention is not intended to limit the scope of the present invention, which is limited only by the following: sea ​​bream.

[0024] As used in this specification and the appended claims, the singular forms "a," "an," and "an" are used interchangeably. It is noted that "the" includes plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes such Reference to "the composition" includes a plurality of peptides. Reference is made to one or more of these compositions and equivalents thereof known to those skilled in the art.

[0025] As used herein, "treating" means preventing or treating the effects of a disease or condition. to slow or partially or completely reverse the effects of the disease or condition To do so, a human or other mammal (e.g., an animal model) having the disease or condition may be used. In some embodiments, the disease or condition is treated by administering a composition of the present invention to a subject. The condition may be a disease or condition related to the CNS, or a CNS disorder or injury. Treatment is intended to reduce the risk of developing pathologies associated with a disease, disorder, and / or condition. Therefore, subjects who do not show symptoms of the disease, disorder, and / or condition, and / or those who have the disease It may also be performed on subjects who show only early signs of a disorder and / or condition. In some embodiments, treatment involves delivery of one or more of the disclosed compositions to a subject. Includes:

[0026] As used herein, "prevent" means to reduce the susceptibility to developing a disease, disorder, or condition. Minimizing the likelihood that a subject with increased size will develop the disease, disorder, or condition. means.

[0027] As used herein, the term "subject" refers to the target of administration, e.g., a human. Thus, the subject of the disclosed method may be a mammal, a fish, a bird, a reptile, or an amphibian, etc. The term "subject" also refers to domestic animals (e.g., cats, dogs, etc.). , livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice In one embodiment, the subject is a mammal. In another embodiment, the subject is a human. The term does not denote a particular age or sex. Therefore, regardless of whether they are male or female, adults, children, It is intended to cover adolescent and neonatal subjects, as well as fetuses.

[0028] As used herein, the term "patient" refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects. In some embodiments, a "patient" is someone who has been diagnosed with a need for treatment prior to the administering step. In some embodiments, patient and subject can be used interchangeably. Cut.

[0029] As used herein, the term "amino acid sequence" refers to the abbreviations that represent amino acid residues. , refers to a list of letters, characters or words. The conventional one-letter code for α, β, β-amyloid, is represented as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, Asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine V, valine; W, tryptophan; Y, tyrosine.

[0030] As used herein, "polypeptide" refers to any peptide, oligopeptide, or polypeptide. A polypeptide refers to a peptide, gene product, expression product, or protein. The term "polypeptide" refers to a naturally occurring or synthetic molecule. Contains.

[0031] Additionally, as used herein, the term "polypeptide" refers to a peptide connected to each other by a bond or modified peptide bond (e.g., peptide isostere) It refers to amino acids, and may contain modified amino acids other than the 20 genetically encoded amino acids. Polypeptides may be synthesized by natural processes, such as post-translational processing, or by other processes known in the art. The peptide may be modified by any of the known chemical modification techniques. In the case of any polypeptide containing an amino acid side chain, and an amino or carboxyl terminus, The same type of modification may occur to the same degree at several sites in a given polypeptide. Modifications may be present in varying degrees, and a given polypeptide may contain many types of modifications. Modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, acetylation, and acetylation. amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of heme moiety, nucleic acid covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative; Covalent binding of phosphitidylinositol, disulfide bond formation, demethylation, cysteine formation of hydroxybenzoates or pyroglutamates, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, and cleavage selenoylation, sulfation, and cleavage of proteins Transfer RNA-mediated addition of amino acids, such as arginylation. roteins -Structure and Molecular Properties ies 2nd Ed.,TECreighton,WHFreeman an d Company, New York (1993), Posttranslation al Covalent Modification of Proteins,BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

[0032] As used herein, the phrase "nucleic acid sequence" refers to DNA or RNA, or DNA -RNA hybrid, single-stranded or double-stranded, sense or antisense hybridizes to complementary nucleic acids by Watson-Crick base pairing regardless of Naturally occurring or synthetic oligonucleotides or polynucleotides that can be The nucleic acid sequences of the present invention may contain nucleotide analogs (e.g., BrdU), and non-nucleotides. Phosphodiester internucleoside linkages (e.g., peptide nucleic acids (PNA) or thiodiesters) In particular, nucleic acid sequences include, but are not limited to, DNA, RNA, cDNA, and may include DNA, gDNA, ssDNA, dsDNA, or any combination thereof .

[0033] As used herein, an "effective amount" of a composition is an amount sufficient to provide a desired effect. The exact amount required will vary depending on the species, age, and the patient's general condition, the severity of the disease (or underlying genetic defect) being treated, the specific The exact "effective amount" will vary from subject to subject depending on the compound, its mode of administration, etc. However, an appropriate "effective amount" can be determined using only routine experimentation. and can be determined by one skilled in the art.

[0034] As used herein, "selectively binds" refers to binding to a nucleic acid sequence (e.g., cargo) Alternatively, the MTS may recognize and physically interact with its target (e.g., a specific cell type) and / or induce other This means that they do not significantly recognize or interact with the target.

[0035] The term "percent homology" is used herein to mean "percent identity" ) and are used interchangeably with sequence alignment programs. nucleic acid or amino acid sequence identity when aligned with the wild-type sequence or the sequence of interest For example, as used herein, 80% homology refers to a level of homology within the defined range. This means the same as 80% sequence identity as determined by the algorithm, and therefore Sequence homology has greater than 80% sequence identity over the entire length of the given sequence. Exemplary levels of sequence identity include, but are not limited to, those found in a given sequence, e.g., 80, 85, 90, 95, 98% or more of any of the MTS sequences set forth in It can be used to determine identity between two sequences. Exemplary computer programs that can be used include, but are not limited to, the BLAST suite of programs. programs, e.g., BLASTN, BLASTX, publicly available on the Internet; and TBLASTX, BLASTP, and TBLASTN. hul, et al.,1990 and Altschul,et al.,199 See also 7. Sequence searches are typically performed using GenBank DNA Sequence Search. When evaluating a given nucleic acid sequence against sequences in the .es and other public databases The BLASTN program is used to search for the BLASTX gene. Amino acids in ank Protein Sequences and other public databases For sequences, it is preferred to search for nucleic acid sequences translated in all reading frames. Both BLASTN and BLASTX have an open gap penalty of 11.0. and extended gap penalty 1.0. Use the SUM-62 matrix (e.g., Altschul, SF, et al., Nucleic Acids Res. 25:3389-3402, 1997. (See, e.g., 1997). A preferred alignment of selected sequences is a "synchronization" between two or more sequences. To determine the "uniqueness" percentage, for example, an open gap penalty of 10.0 and an extended gap penalty of 10.0 are used. The default parameters included a 0.1 drop penalty and a BLOSUM 30 similarity matrix. CLUSTA on Mac Vector version 13.0.7 operated using the This is done using the LW program.

[0036] Substitutions, deletions, insertions, or any combination thereof may be used to create the final derivative, variant. Generally, these modifications are performed by minimizing the modification of the molecule. To minimize this, it is performed on several nucleotides. In some cases, larger variations may be tolerated.

[0037] Generally, the nucleotide identity between individual variant sequences is at least 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Thus, a "variant sequence" is a sequence that is different from a parent or reference sequence of the present invention (e.g., The sequence may have a specific identity to the parent sequence (wild-type sequence), including, but not limited to, Specificity and / or activity of the sequence is at least 80%, 81%, 82%, 83%, 84%, 85% %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 96%, 97%, 98%, or 99% of the DNA share a common biological function. For example, A "variant sequence" is a sequence that has one, two, three, or four amino acid residues in comparison to a parent or reference sequence of the present invention. Contains nucleotide base changes and shares or may share the biological function, specificity and / or activity of the parent sequence. Thus, a "variant sequence" may be a sequence that is an improvement over a parent sequence of the present invention. The specific identity of the parent sequence may be, but is not limited to, a specific identity of the parent sequence and / or are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, and 87% active ,88%,89%,90%,91%,92%,93%,94%,95%,96%,97% 98%, or 99%, share biological function. Variant sequences also may be used interchangeably with a reference sequence. At least 80%, 81%, 82% of the specificity and / or activity of the sequence (e.g., MTS sequence) ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% can be shared do.

[0038] "Optional" or "optionally" refers to the event, circumstance, or material described thereafter. may or may not occur, may or may not exist, and The description of the event, circumstance, or material occurs or exists, and This means that it includes cases where the value does not occur or does not exist.

[0039] Ranges are expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, the context may dictate otherwise. Unless otherwise stated, ranges from one particular value and / or to another particular value are not specified. Similarly, when values ​​are expressed as approximations, In these cases, the use of the antecedent "about" does not specifically imply a particular value unless the context indicates otherwise. Unless otherwise specified, the present invention forms another specifically contemplated embodiment that should be considered disclosed. It is understood that the endpoints of each of the ranges are It is further understood that a given value is significant in relation to the other endpoint and independently of the other endpoint, unless otherwise specified. Finally, individual values ​​and subranges of values ​​that fall within the explicitly disclosed ranges will be are specifically contemplated and, unless the context otherwise indicates, It should be understood that the above should be considered as a disclosure. In such cases, whether or not some or all of these embodiments are explicitly disclosed. This rule applies regardless of the

[0040] Unless otherwise defined, all technical and scientific terms used herein are disclosed. The terms "compounds" and "methods" have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains. Any methods and materials similar or equivalent to those described herein may be used in the methods of the present invention. Although particularly useful methods, devices, and materials may be used in the practice or testing of the methods and compositions described herein, Publications cited herein and the material for which they are cited are incorporated by reference. Nothing herein shall be construed as constituting a license to any of the foregoing. shall not be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of the literature states what its authors assert, and applicants must provide accurate information on the accuracy of the documents cited. The right to dispute gender and relevance is reserved. Several publications are referenced herein. However, such references should not be construed as a guarantee that any of these documents constitutes part of the general knowledge in the art. It should be expressly understood that this does not constitute an acknowledgment that the

[0041] Throughout the description and claims of this specification, the word "comprises" The word "comprising" and variations of this word, such as "comprising" and "including" "(comprises)" means "including, but not limited to," e.g. , and is not intended to exclude other additives, ingredients, integers or steps. In any method described as comprising one or more steps or actions, each step is It is specifically intended to include steps that "consist of" or "are" (Except when the step contains a specific term), this means that each step is is not intended to exclude other additives, ingredients, integers, or steps not listed. This means:

[0042] B. Peptides It can provide molecular transport or can be targeted to a specific location or site. The peptides that can be produced can be called MTS peptides. or site and thus be able to deliver cargo to that specific location or site. In some embodiments, the amino acid sequence is known as a molecular transport system. The MTS peptides disclosed herein target the CNS.

[0043] MTS peptides or targeting peptides are disclosed herein. Disclosed is an MTS peptide containing any of the amino acid sequences set forth in sequences 1 to 10. In some embodiments, the MTS peptides are used to transport molecules to specific locations or sites. or an amino acid sequence responsible for targeting. For example, in some embodiments, the disclosed The MTS peptides are those having any of the sequences set forth in SEQ ID NOs: 1 to 10, as shown in Table 1. It consists of an amino acid sequence. [Table 1]

[0044] In some embodiments, one or more MTS peptides are set forth in SEQ ID NOs: 1-10. 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity. The S peptide may be at least 90, 91 or 100% covalently linked to any of the MTS peptides disclosed herein. , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity In some embodiments, one or more MTS peptides comprise at least one of the following in the active portion of the peptide: The active portion of the peptide has 100% identity with the CSF, allowing passage from the blood into the CSF. Thus, in some embodiments, the MTS peptide and at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the identity occurs outside the active portion.

[0045] In some embodiments, the MTS peptide may be modified. Modifying the MTS peptide can optimize or stabilize the peptide. This includes:

[0046] In some embodiments, the MTS peptides are prepared by the method of claim 1, wherein the MTS peptides are substituted with the MTS peptides during synthesis and / or storage. can be stabilized so that the In this embodiment, the MTS peptide comprises the sequence DGYKLQTSLDWQMWNP (SEQ ID NO: 2). The peptide replaces the glycine at the second amino acid position with DAYKLQTSLDWQMWNP (sequence It can be stabilized by exchanging it for an alanine such as number 1, and the N-terminal acetyl group In some embodiments, aspartic acid and The dipeptide of glycine (DG in SEQ ID NO: 2) can form a cyclic intermediate (Figure 19). The cyclic intermediate can then be opened again to give the original MTS peptide or a non-natural amino acid. This can result in the formation of amino acids and therefore less stable MTS peptides.

[0047] In some embodiments, the MTS peptides may be optimized. Optimized Peptides can be obtained by applying modifications to the individual parent peptide sequences. Use the nucleotide sequence to identify essential amino acids in the parent sequence required for passage from blood to CSF. These modifications can be made by altering the amino- and c-terminal regions of the parent peptide. PEG11 can be obtained by a combination of Ninscanning and cleavage. S provides protection for the C-terminus of the peptide and connects the peptide with a cysteine ​​at the C-terminus. It provides a spacer between the peptide and the covalent bond, improving the solubility of the MTS peptide. Acetylation (CH3CO-) and / or amino acid synthesis with d-amino acids such as d(Leu) The terminal modifications can protect against degradation by peptidases in the blood. There is no uniform length of optimized peptides applicable to all MTS peptides, and all variations The cleavage can be tested to determine the effect on peptide uptake and stability.

[0048] In some embodiments, the MTS peptides disclosed herein have an N-terminal protecting group. In some embodiments, the N-terminal protecting group is a group that allows a protease to selectively bind the amino acid sequence from the N-terminus to the amino acid sequence. In some embodiments, the present invention may be any that prevents cleavage of the The disclosed MTS peptides can be modified by acetylation on the N-terminus. In embodiments, the N-terminal protecting group is an acetyl group. Thus, the MTS peptides disclosed herein can be acetylated. In this case, the N-terminal protecting group may be, but is not limited to, PEGylation, formyl, CH3-(CH)nC O, fluorophores, fatty acids, alkylamines, sulfonamides, or carbamates In some embodiments, the MTS peptides disclosed herein can be derived from the nucleic acid sequence In some embodiments, the chemical conjugate The conjugate may be polyethylene glycol (PEG). In some embodiments, the MTS peptides disclosed herein can be PEGylated. wherein the number of PEG units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or In some embodiments, the number of PEG units is greater than or equal to one or more MTS units. To prevent any steric interference between the peptide and the cargo, one or more MTS peptides The length of the ion exchange membrane may be sufficient to separate the ion exchange membrane from the cargo. Therefore, the MTS peptides disclosed herein can further comprise a linker. The terms "antibody" and "chemical conjugate" can be used interchangeably. In embodiments, the linker is on the C-terminus of the MTS peptide. a composition comprising a chemical conjugate or linker, wherein the chemical conjugate or linker is PEG; and the PEG comprises 11 PEG units. The MTS peptides disclosed herein are those having the sequences set forth in SEQ ID NOS: 1 to 10. and SEQ ID NOs: 1 to 10 may be acetylated on the N-terminus. , may be chemically conjugated to PEG, and the cargo, e.g., a nucleic acid sequence, In some embodiments, the N-terminal protecting group is a D-amino group. The amino acid may be an artificial amino acid such as guanine.

[0049] In certain embodiments, the MTS peptides disclosed herein can be truncated. In some embodiments, the MTS peptides disclosed herein may comprise an active portion of the MTS peptide. In some embodiments, the MTS peptide is truncated to remove all amino acids except Active portions of thiazol-1,2-dione and thiazol-1,2-dione can be used in the disclosed compositions and methods. In this embodiment, the active moiety can be isolated by techniques well known in the art, e.g., alanine scanning The active portion of the MTS peptide can be determined using a cleavage assay or a cleavage test. For example, SEQ ID NO: 7 has at the N-terminus In some embodiments, SEQ ID NO: 7 is C It may be truncated by up to 8 amino acids on the end. The sequence YSMNAQRQHERS (SEQ ID NO: 11) is the active portion of SEQ ID NO: 7. In some embodiments, the amino acid sequence YSMN (SEQ ID NO: 12) is an active portion of SEQ ID NO: 7. Thus, in some embodiments, SEQ ID NO: 12 is used as an MTS peptide. It can be used.

[0050] In some embodiments, stabilized variants of the MTS peptides disclosed herein include It will be disclosed.

[0051] Also disclosed are peptides comprising an MTS peptide conjugated to a cargo. In some embodiments, two or more MTS peptides may be conjugated to the cargo. can.

[0052] A peptide comprising a first MTS peptide conjugated to a cargo, The MTS peptide contains any of the amino acid sequences set forth in SEQ ID NOs: 1 to 12. Peptides are disclosed.

[0053] In some embodiments, the cargo molecule may be, but is not limited to, a nucleic acid sequence, a protein, an antigen It may be an antibody, peptide, nanoparticle, dye, chemical compound, or small molecule. As described above, the cargo is a nucleic acid sequence. In some embodiments, the cargo is an imaging agent, It may be a radionuclide or a detectable marker.

[0054] a first MTS peptide conjugated to a cargo, and a second MTS peptide For example, a peptide is disclosed, further comprising a first M conjugated to a cargo. A peptide comprising a TS peptide, wherein the first MTS peptide is any of the peptides set forth in SEQ ID NOs: 1 to 12. A peptide comprising the amino acid sequence of any of the sequences described above, and further comprising a second MTS peptide. Chid is disclosed.

[0055] In some embodiments, the second MTS peptide is the same as the first MTS peptide. In some embodiments, the second MTS peptide is different from the first MTS peptide. do.

[0056] The attachment of MTS to the cargo can be achieved via maleimide, click, or amide chemistry. The cargo can be attached via a cleavable linker or a non-cleavable linker. Possible linkers can be used to connect them.

[0057] MTS dimers can be synthesized using linear peptide chemistries such as FMOC (which does not contain a maleimide group). The cargo can then be synthesized using maleimide chemistry, click chemistry, amidation, The dimers can be connected by amines and by hydrazones.

[0058] Examples of how to make the conjugates can be seen in Figures 28 and 29.

[0059] C. Composition Disclosed are compositions comprising one of many of the disclosed peptides. In one embodiment, compositions comprising one or more of the MTS peptides disclosed herein are disclosed. A composition comprising a peptide, wherein the peptide is a first peptide conjugated to a cargo. Also disclosed are compositions comprising the MTS peptides of the formula:

[0060] For example, a composition comprising a peptide, wherein the polypeptide is conjugated to a cargo. The first MTS peptide is selected from the group consisting of: Disclosed are compositions comprising the amino acid sequence of any of the sequences disclosed.

[0061] 1. Pharmaceutical Compositions In some embodiments, the disclosed compositions can be pharmaceutical compositions. In some embodiments, a nucleic acid sequence conjugated to one or more MTS peptides is A pharmaceutical composition is disclosed, comprising a composition comprising the compound of formula (I) and a pharmaceutically acceptable carrier. "Acceptable to the target" means, as known to those skilled in the art, to minimize any degradation of the active ingredient and It means a material or carrier selected to minimize any adverse effects on the Examples include dimyristoyl phosphatidylcholine (DMPC), phosphate buffered saline, or Examples of liposomes include multivesicular liposomes. For example, PG:PC:cholesterol:peptide or PC:peptides can be used as carriers in the present invention. Acceptable carriers and their formulations are described in Remington: The Science and d Practice of Pharmacy(19th ed.)ed.ARG ennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of pharmaceutical agent is added to render the formulation isotonic. Pharmaceutically acceptable salts are used in the formulation. Other examples of pharmaceutically acceptable carriers include, but are not limited to, Examples of solutions that are not commonly used include saline, Ringer's solution, and dextrose solution. H can be about 5 to about 8, or about 7 to about 7.5. Sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the active ingredient. The materials are used in shaped articles such as films, stents (those implanted in blood vessels during angioplasty), The particular carrier may be in the form of a liposome, a microparticle, or a carrier that depends, for example, on the route and dosage of administration. It will be apparent to those skilled in the art that depending on the concentration of the composition used, different concentrations may be more suitable. These are most typically solutions such as sterile water, saline, and buffers at physiological pH. These are standard carriers for drug administration to humans.

[0062] Pharmaceutical compositions also include those intended for the polypeptides, peptides, or conjugates of the invention. The composition may contain carriers, thickeners, diluents, buffers, preservatives, etc., as long as the activity of the composition is not impaired. Pharmaceutical compositions can also contain one or more active ingredients, such as antibacterial agents, anti-inflammatory agents, anesthetics, etc. In addition to the above composition.

[0063] The pharmaceutical compositions disclosed herein can be prepared for oral or parenteral administration. Pharmaceutical compositions prepared for parenteral administration include intravenous (or intraarterial) and intramuscular. Intrameatal, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), or transdermal (e.g., For example, those prepared for topical administration. The composition may also deliver proteins. Acceptable carriers include, but are not limited to, aqueous carriers such as buffered saline (e.g., PBS). The composition may be formulated for parenteral administration containing the fusion protein dissolved or suspended in a suitable carrier. One or more of the excipients included may be pH adjusters and buffers, osmolality adjusters, wetting agents, detergents, etc. This may be useful for approximating physiological conditions such as those of a drug. When a solid component is included (e.g., a tablet, capsule, etc.), one or more of the excipients may be The composition can act as a binder or filler (for formulations of the present invention). When formulated for application to a membrane surface, one or more of the excipients may be a cream, ointment, or It may be a solvent or emulsifier for the formulation of the above.

[0064] The pharmaceutical compositions may be sterile, or may be sterilized by conventional sterilization techniques. Aqueous solutions, either neat or lyophilized, are encompassed by the present disclosure. The drug may be packaged as a lyophilized preparation which is then incorporated into a sterile aqueous carrier prior to administration. The pH of the pharmaceutical composition is typically between 3 and 11 (e.g., about 5 to 11). 9), or 6 to 8 (e.g., about 7 to 8). The resulting composition in solid form may contain multiple units. It can be packaged in single dose units, for example, sealed packages of tablets or capsules. Each of the solid forms contains a certain amount of one or more of the above-mentioned drugs. Flexible tubes, such as squeezable tubes designed for topically applied creams or ointments It may be packaged in a container for the amount.

[0065] The pharmaceutical compositions described above are formulated to contain a therapeutically effective amount of the compositions disclosed herein. In some embodiments, therapeutic administration encompasses prophylactic applications. Based on these and other prognostic methods, physicians may, in consultation with their patients, The patient has a clinically determined predisposition or high susceptibility to one or more autoimmune diseases. If the patient has a high susceptibility (in some cases, a very high susceptibility) or if the patient has a high risk of developing cancer Clinically determined predisposition or high susceptibility (in some cases, extremely high susceptibility) In cases where the patient has a high risk of developing the disease, prophylactic administration may be selected.

[0066] The pharmaceutical compositions described herein can be administered to a subject (e.g., a human subject or human patient) to treat a clinical disease. The compound can be administered in an amount sufficient to delay, reduce, or preferably prevent the onset of the disease. Thus, in some embodiments, the subject is a human subject. In this regard, the composition may be administered to a subject already suffering from or diagnosed with a CNS disease or disorder (e.g., and administering to a subject (e.g., a human subject) at least partially ameliorating a sign or symptom of the condition or causing the condition to worsen. an amount sufficient to inhibit (preferably stop) the progression of the symptoms, their complications, and consequences of An amount adequate to accomplish this is defined as a "therapeutically effective amount." A therapeutically effective amount of a composition may be an amount that achieves a cure, although that result may be achieved in any number of ways. As mentioned above, a therapeutically effective amount is a dose that is sufficient to prevent the onset or progression of cancer. Slowing, hindering, or preventing a CNS disease or disorder, or CN S includes an amount that provides treatment in which the symptoms of a disease or disorder are alleviated. The severity of the condition may be less, and recovery can be accelerated in treated individuals.

[0067] The total effective amount of the conjugates in the pharmaceutical compositions disclosed herein may be administered as a single dose. The compound may be administered to a mammal as a bolus or by infusion over a relatively short period of time. or a fractionated treatment protocol where multiple doses are given over a longer period (e.g. For example, one dose every 4 to 6, 8 to 12, 14 to 16, or 18 to 24 hours, or two or more doses every 4 to 6, 8 to 12, 14 to 16, or 18 to 24 hours. can be administered using a dose of every 4 days, every 1-2 weeks, or once a month Alternatively, continuous intravenous infusion sufficient to maintain a therapeutically effective concentration in the blood is also within the scope of this disclosure. It is inside.

[0068] D. Method Using one or more of the disclosed peptides (e.g., MTS peptides) or compositions, Disclosed are methods for using the peptides (e.g., MTS peptides) or compositions disclosed herein. Any of the compositions can be used in the methods disclosed herein. In some embodiments, the MTS peptide conjugated to a cargo is delivered from the blood. can pass from the vasculature to the CNS.

[0069] 1. How to transport cargo A method for transporting cargo to the CNS, comprising administering one or more of the disclosed compositions to administering to a subject in need thereof an MTS peptide conjugated to a cargo. In some embodiments, the conjugated cargo enters the CNS. The conjugated MTS peptide enters the choroid plexus. The conjugated MTS peptides enter the cerebrospinal fluid (CSF). In this context, CSF transports cargo-conjugated MTS peptides throughout the CNS. In some embodiments, the MTS peptide is cleaved from the cargo and released into the CSF. It transports cargo via the ATP and can be separated from the MTS peptide throughout the CNS.

[0070] In the disclosed methods, the cargo, upon delivery to the CNS, exhibits functional activity within the CNS. Hold.

[0071] In some embodiments, the administration is intravenous. Intravenous (or intraarterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal), or transcutaneous In some embodiments, administration is via aerosol inhalation. This can be done.

[0072] 2. Treatment methods A method of treating a CNS disorder or injury, comprising administering a disclosed peptide (e.g., MTS peptide) to a subject. administering one or more of the following to a subject in need thereof: Disclosed are methods wherein the cargo is a therapeutic agent for treating a CNS disorder or injury.

[0073] In some embodiments, the CNS disorder or injury includes, but is not limited to, Parkinson's disease, Alzheimer's disease, glioblastoma and other cancers that metastasize to the brain, amyotrophic lateral sclerosis, multiple sclerosis Thus, in some embodiments, the CNS disorder may be a neurodegenerative disorder, a neurodegenerative disorder, or a traumatic brain injury. Therapeutic agents for treating the injury or damage include, but are not limited to, antibodies, gene therapy, chemical compounds, nucleic acid sequences, or peptides. In some embodiments, the CNS disorder or injury may be a CNS peptide (or protein). Specific examples of therapeutic agents include, but are not limited to, N-methyl D-aspartate (NMDA) acetate. antagonists, chemotherapeutic agents, glutamate antagonists, or immunomodulators (e.g., immunosuppressive or immunoactivating factors).

[0074] 3. How to capture images 1. A method of imaging the CNS, comprising administering to a subject a disclosed peptide (e.g., an MTS peptide) or and administering one or more of the compositions to a subject in need thereof, wherein the cargo is As used herein, imaging agent, imaging label, or imaging An imaging moiety (also a detectable label, detectable moiety, or diagnostic moiety) can be an atom, molecule, or refers to an atom, molecule, or composition whose presence can be measured directly or indirectly. vinegar.

[0075] In some embodiments, the imaging agent may be, but is not limited to, a fluorescent dye, a radioisotope, a magnetic Can be beads, metal beads, colloidal particles, near-infrared dyes, or electron-dense reagents Thus, detectable moieties include, but are not limited to, fluorescent moieties, radioactive moieties, or electronic moieties. In some embodiments, the imaging agent comprises two moieties, moiety 1 being moiety a disease-specific peptide, nucleic acid, or compound conjugated to moiety 2; are fluorescent dyes, radioisotopes, magnetic beads, metal beads, colloidal particles, near-infrared dyes, or an electron-dense reagent. In some embodiments, portion 1 of the imaging agent is and portion 2 of the imaging agent is attached to a disease-specific indicator for detection or visualization of the imaging agent. In some embodiments, a disease-specific peptide, nucleic acid, or compound Therapeutic uses include, but are not limited to, Parkinson's disease, Alzheimer's disease, glioblastoma, and other tumors that metastasize to the brain. Indicators of certain diseases such as cancer, amyotrophic lateral sclerosis, multiple sclerosis, and traumatic brain injury It can bind to certain CNS proteins or nucleic acids. Binding of disease-specific peptides, nucleic acids, or compounds to proteins or nucleic acids within the NS can be detected or visualized by part 2 of the imaging agent.

[0076] The disclosed methods of imaging the CNS are useful for monitoring the CNS for the presence of disease or disease progression. For example, the presence of a disease or disease progression can be detected. Upon receiving the peptide, a second MTS peptide conjugated to a cargo is administered to the subject. and the cargo is a therapeutic agent. In some embodiments, the first peptide is The cargo comprises an MTS peptide conjugated to a medicament for detecting the presence of glioblastoma in the CNS. If the imaging agent is to be identified, the second peptide may be conjugated to a cargo, such as MTS. The cargo may comprise a peptide, and is a therapeutic agent specific for glioblastoma.

[0077] Also provided is a method of imaging the CNS, comprising: and administering a peptide comprising one or more of the disclosed MTS peptides. wherein the first cargo is an imaging agent and the second cargo is a therapeutic agent for treating a CNS disorder or injury. Thus, in some embodiments, imaging the CNS is If the presence of disease or disease progression is detected, a therapeutic agent for a CNS disorder or injury can be administered. It can provide a therapeutic effect.

[0078] E. Kit The above-mentioned and other materials may be used in kits or as a means to carry out the disclosed methods. or any suitable combination as a kit useful for aiding in the practice of the disclosed methods. The kit components in a given kit may be packaged together in a single package for use in the disclosed methods. For example, the disclosed peptides are useful when designed and adapted for use with Disclosed are kits comprising one or more of the methods. [Example]

[0079] DiaCyt delivers a wide variety of therapeutic cargoes to the CN without physically disrupting the BBB. This is a new platform technology for identifying delivery agents that selectively transport to S. A selection platform has been developed to identify peptide-based molecular transport systems (MTS). , which can pass through the CNS without disrupting the barrier and can protect against antibodies, proteins, and therapeutic agents containing biomolecules such as nucleic acids, without inactivating them during transport into the CNS. can deliver MTS-cargo to the CNS, release MTS-cargo, and distribute the cargo throughout the CNS do.

[0080] This concept is unique in two ways. First, selection has the key characteristics mentioned above. This is an unbiased phenotypic approach that allows for the selection of MTS. Identify MTS that can cross the CNS barrier and be released into the CNS. Therefore, the identified MTS is required for the transport of functional bacteriophage. They transport biological materials through cells without degrading the cargo during the process.

[0081] The second unique feature is that the choroid plexus is targeted as opposed to the endothelial BBB. This is based on the physiological structure and biological functions of the choroid plexus (see Appendix). This is advantageous in overcoming the barrier of delivery through the endothelial BBB, which has been a focus of attention in the field. The capillaries are fenestrated (leaky) and have astrocytic foot processes around the vessels. It is not permeable to blood and allows materials outside of blood to pass through.

[0082] Figure 1 shows that the BBB is a powerful biological barrier. C through the intercellular gap Access of drugs and biological therapeutics to NS tissues is not an option. The peptides, compositions, and methods described provide a new paradigm for drug development. The choroid plexus produces cerebrospinal fluid (CSF). Epithelial cells form the blood-cerebrospinal fluid barrier (BCSFB). Unlike the BBB, capillaries have fenestrations (endothelial gaps between cells), allowing immune cells, proteins, and even pathogens to escape Choroid plexus epithelial cells transport ions, peptide hormones, and proteins to the blood. Tight junctions are specialized cells that transport fluid from the pleural effusion to the CSF. It is absent from the limiting membrane, allowing paracellular diffusion between the CSF and the brain.

[0083] Figure 2 shows a schematic diagram of capillaries in the choroid plexus and how they transport cargo to the CN. Some of the structural advantages of targeting the choroid plexus are: ) The capillaries of the choroid plexus are fenestrated (leaky), allowing outside materials to pass through the blood 2) the absence of astrocyte foot processes around blood vessels Several biological advantages of targeting the choroid plexus include the ability to target molecules, peptides, and proteins within the CNS. The goal is to take advantage of the natural role of choroid plexus epithelial cells in transporting proteins and cells. Another biological advantage of targeting the plexus is the transport of cargo into the CSF and then throughout the CNS. The key is to circulate it.

[0084] 1. For Unbiased Selection to Identify MTS Peptides Mediating Functional Transport Activity One experimental design is shown in Figure 3. The identified MTS can be used for cargo delivery to the CNS. This can be easily optimized by chemical means to generate MTS suitable for can.

[0085] One experimental design for validation of in vivo delivery of MTS to the CNS is shown in Figure 4. The design mediates functional transport across cell barriers via a pathway that protects cargo from degradation. Allows you to select MTS.

[0086] Figure 5 outlines how the system for identifying MTS peptides was developed. This system transcytoses in an in vitro model and C in an in vivo model. One MTS phage clone was identified that had the ability to enter SF. Synthetic MTS_CPE Data using C4 (DGYKLQTSLDWQMWNP (SEQ ID NO: 2)) were obtained in vitro. demonstrated in vitro and in vivo activity.

[0087] Figure 6 shows tissue staining of the CPEC4 phage clone in the choroid plexus and ventricles, and Figure 7 Figure 1 shows tissue staining of the CPEC4 phage clone outside the ventricular system in the brain. delivers MTS-cargo to the CNS, potentially allowing transport of the cargo to other regions of the brain. The phage clones were injected intravenously via the tail vein of rats. After allowing the phage clones to circulate for a period of time, the animals were perfused terminally and the brains were isolated. The brains were sectioned and processed for immunohistochemistry using an anti-M13 phage primary antibody. The blue staining is nuclear staining. Phage staining was observed in the ventricles and surrounding tissue. This was observed in the CSF, indicating the transport of the phage clone within the CSF.

[0088] These data suggest that primary human choroid plexus epithelial cells replicate the tight junctions found in the human choroid plexus. An in vitro model of the choroid plexus was established for use in the study. A rat model was also developed. Selection for identifying phage clones from the library that cross the endothelial barrier was also performed. The peptide is synthesized outside the phage environment and the peptide exerts its transport activity. showed that it can hold and transport small molecules and proteins across the barrier. The data show that the MTS peptide selectively transports the glycine into the CSF. This confirmed the sending of the

[0089] Once MTS peptides have been identified, they can be optimized by several means, including chemical modification. Optimization can be performed to improve solubility, stability, brain distribution, biodistribution, cellular transport, The desired result of using MTS is that it can increase the circulation time of CN. At least 2.5% of the injected dose is present in the S, but 1.5% of the injected dose is present in the CNS. Greater than 5%.

[0090] The current study was performed in vitro using Transwell screened and validated assays. The approach was used to provide targeting in animal models and to assess delivery efficiency (real-time CSF analysis, histology, radiolabeling and / or ex vivo analysis of CSF with imaging) We investigated the effect of various cargoes (peptides, proteins, and nucleic acids) on MTS delivery efficiency. ) to evaluate.

[0091] Figure 10 shows an unbiased selection process to identify MTS peptides that mediate functional transport activity. For this purpose, we used a monolayer of rat choroidal epithelium (Z310 cells) in a Transwell bucket. Figure 1 shows a schematic diagram of the experimental design. Z310 cells were cultured in the ventricles (CSF side) after incubation. The transported phage present on the basal side (the blood side of the choroid plexus) was used to After this incubation, the cells were exposed to the phage library. Phages were also recovered from the lysate. The library used in this selection was a random library of synthetic codons. The 16-mer peptide generated by the addition was expressed.

[0092] Figure 11 shows the results from Transwell and lysate samples in panning rounds 4 and 5. Sequences obtained from 96 individual phage clones submitted for DNA sequencing Among them, the phage clone encoding the MTS_CPEC4_V1 peptide sequence (SEQ ID NO: 2) MTS_CPEC4_V1 is 2.6 × 10 10 First This peptide was selected from a random 16-mer peptide library of high complexity. The clone was the dominant sequence observed in panning rounds 4 and 5 of the selection. The frequency of observing this particular sequence in the output from rounds 4 and 5 is shown in Figure 11. As shown, this arrangement allows for the transport of power from the Transwell, and The antibodies were observed in both cell lysates at the end of the incubation period. The total number of CPEC4_V1 clones observed in outputs of bands 4 and 5 was 365 sequences. The total number of nucleotides was 144. Only one other peptide was observed, but at a much lower frequency (6 / 365).

[0093] Transwell selection was completed on human primary choroid plexus epithelial cells to obtain the lead peptide. (Figure 12) CPEC4_phage clone sequence DGYKLQTSLDWQMWNP( SEQ ID NO: 2) was the lead peptide in these studies. The CPEC4 phage clone was enriched by the PCR amplification. Clones are selected from transwells that demonstrate transport and release. CPEC4 phage clones can be seen in cell lysates, which indicates that the This indicates no active transport.

[0094] FIG. 12 shows that the CPEC4 peptide (SEQ ID NO: 2) induces large cross-sectional migration across the choroid plexus epithelial cell layer. The phage clones were shown to mediate the transport of phage particles. It remains the same.

[0095] Figure 13 shows a dimer of the CPEC4 peptide. The CPEC4 peptide is a dimer. , as having a biotin handle that allows for the attachment of fluorescently labeled proteins. It was done.

[0096] Figure 14 shows that MTS_CPEC4_V1 (SEQ ID NO: 2) inhibits the growth of human coronary arteries in an in vitro model. We show that 6-9% of the proteins mediate the transport of fluorescent molecules across the cell plexus. At the end of the experiment, the lack of dextran transport and the maintenance of transepithelial resistance Tight junctions remain intact as evidenced by

[0097] Figure 15 shows that the MTS_CPEC4 phage clone or peptide binds to rat (Z310 cells) Used to determine the ability of cells to cross the choroid plexus barrier model derived from the cytoplasmic membrane of the in vitro Transwell assay. The amount of input placed on one side of the barrier (the "blood" side), and The amount of phage crossing the cells to the other side (CSF side) is measured. The data for the empty phage are shown. The amount of phage on each side of the well is determined by bacterial titration. The bottom table presents data for synthetic peptides, where the peptides are The peptides on either side are labeled with a fluorophore so that they can be measured. A trans dye (such as Alexa Fluor 488) is used as a control molecule. The child does not pass through the Transwell, ensuring that the choroid plexus model is valid. .

[0098] Figure 16 illustrates how the MTS delivery system targets the CNS. After intravenous injection, CSF can be removed from the brain by capillary puncture of the cisterna magna, and phage The existence of

[0099] FIG. 17 shows that the CPEC4 phage clone preferentially binds to CSF ​​compared to the control phage. and therefore, are able to mediate transport into the CSF. The roan remains alive during transport to CSF.

[0100] Figure 18 shows the accumulation of MTS-CPEC4_V in the CSF of rats after mouse tail vein injection. 2 (SEQ ID NO: 1) shows the experimental design and final results.

[0101] MTS was able to synthesize more than 1 milligram of high-quality monomeric peptide. It has been difficult to synthesize dimeric peptides, and dimeric peptides have been even more difficult. Inserting "TS" in the middle of the MTS_CPEC4_V2 sequence using r dipeptide It was determined that beneficial results were obtained (Figure 20).

[0102] As shown in Figure 21, CPEC4_V1 (SEQ ID NO: 2) and CPEC_V2 (SEQ ID NO: No. 1) is a chemical cargo (AF647 dye) and a protein cargo (50 kDa streptavidin). These antibodies are efficiently transported across a monolayer of choroid plexus cells using either a phosphodiesterase (PTA) or a phosphodiesterase (PTA). The integrity of the monolayer with the adhesive bonds was confirmed by the addition of a low molecular weight dye (750 maleimide), a 10 kDa dye. lack of diffusion or transport of strand polymers or larger proteins (50 kDa) In the SA conjugate, MTS has biotin in the core structure. Biotin is then attached to a fluorophore-labeled streptavidin protein. Direct conjugation involves attaching a fluorophore to an MTS on the core. covalently linked to the antibody (i.e., without using a biotin / streptavidin linkage) means.

[0103] The stability of different MTS sequences is important. Figure 22 shows that MTS_CPEC4_V2 is 80% of the peptide remains intact at 24 hours. The MS data showed that NP-PEGH11 was converted to methionine or This indicates possible oxidation losses of tryptophan.

[0104] The MTS_CPEC4_V2 (SEQ ID NO: 1) dimer was demonstrated in vivo as shown in FIG. MTS is distributed throughout the ventricular system of the brain, which means that MTS , indicating passage from the bloodstream into the CSF. MTS allows visualization of its location. The dye is labeled with a dye and injected intravenously into the tail vein of rats. After the indicated time, CSF is isolated from the cisterna magna. The brain is harvested. After the fixation period, the brain is separated by 1 mm coronal sections. The brain is sectioned into segments (anterior to posterior). Individual sections of the brain are imaged using fluorescence and then peeled. Determine the location of the region of peptide accumulation.

[0105] Other lead MTS peptides were tested using a Transwell transport setup with rat choroid plexus cells. Eight phage clones were enriched in CSF and then tested individually (Figure 24). The phage clones were transcytosed in vitro into Z310 choroid plexus cells. MTS_Z310-2 (SEQ ID NO: 4) was tested for CSF accumulation in mice and rats. ) and MTS_Z310-5 (SEQ ID NO: 7) phage clones were retained for further evaluation. Figure 25 shows that the MTS_Z310-2 and MTS_Z310-5 peptides 20% transport was observed across the choroid plexus epithelial cells. This is better than that seen with CPEC4. Based on alanine scanning, MTS_ The underlined portion of Z310-5 has been identified as the key peptide region responsible for activity.

[0106] Dimer MTS_Z310-5 is present in a three-fold higher amount than MTS_CPEC4_V2 MTS is distributed throughout the ventricular system of the brain, which means that MTS is transported from the bloodstream to the brain. The MTS allows visualization of the location of the blood vessels. The dye is labeled and injected intravenously into the tail vein of rats. After the indicated time, the CSF After the fixation period, the brain is separated into 1 mm coronal segments. Individual brain sections are imaged using fluorescence to determine peptide accumulation. Determine the location of the product region.

[0107] Dimeric MTS_Z310-2 is present in 9-fold higher abundance than MTS_CPEC4_V2 (Figure 27). MTS is then distributed throughout the ventricular system of the brain, indicating that MTS , indicating passage from the bloodstream into the CSF. MTS allows visualization of its location. The dye is labeled with a dye and injected intravenously into the tail vein of rats. After the indicated time, CSF is isolated from the cisterna magna. The brain is harvested. After the fixation period, the brain is separated by 1 mm coronal sections. The brain is sectioned into segments (anterior to posterior). Individual sections of the brain are imaged using fluorescence and then peeled. Determine the location of the region of peptide accumulation.

[0108] The chemical conjugation of the dimer and MTS to the cargo can be seen in FIG. MTS constructs, particularly monomers and dimers, with different reactive groups or chemical probes on the core The general structure of the construct is shown.

[0109] Those of ordinary skill in the art will be able to readily identify, using no more than routine experimentation, the methods and compositions described herein. Many equivalents to the specific embodiments of the present invention will be recognized or can be identified. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims

1. A peptide, DAYKLQTSLDWQMWNP (SEQ ID NO: 1) DGYKLQTSLDWQMWNP (SEQ ID NO: 2), NQEYQHHKIKVRPSHQ (SEQ ID NO: 3), FPSWTSKNQQWTNQRQ (SEQ ID NO: 4), AHMSQKRLPHQVHQHQ (SEQ ID NO: 5), AGNKYEYTMHQKHNK (SEQ ID NO: 6), SKETYSMNAQRQHERS (SEQ ID NO: 7), HRYDADRHHSFTPQYH (SEQ ID NO: 8), NEEMHQAQRHHVQW (SEQ ID NO: 9), or A peptide comprising the amino acid sequence ALEPWGYKQVIKMAPN (SEQ ID NO: 10). 。

2. The peptide of claim 1 , wherein the peptide has an N-terminal protecting group.

3. 3. The peptide of claim 2, wherein the N-terminal protecting group is an acetyl group.

4. The peptide according to any one of claims 1 to 3, wherein the peptide further comprises a linker. 。

5. 5. The method of claim 4, wherein the linker is a polyethylene glycol (PEG) linker. peptides.

6. The peptide of claim 5 , wherein the PEG linker is PEG11.

7. 7. The method of claim 4, wherein the linker is on the C-terminus of the peptide. peptides.

8. Peptides comprising a first molecular transport system (MTS) peptide conjugated to a cargo wherein the first MTS peptide is DAYKLQTSLDWQMWNP (SEQ ID NO: 1) DGYKLQTSLDWQMWNP (SEQ ID NO: 2), NQEYQHHKIKVRPSHQ (SEQ ID NO: 3), FPSWTSKNQQWTNQRQ (SEQ ID NO: 4), AHMSQKRLPHQVHQHQ (SEQ ID NO: 5), AGNKYEYTMHQKHNK (SEQ ID NO: 6), SKETYSMNAQRQHER (SEQ ID NO: 7), HRYDADRHHSFTPQYH (SEQ ID NO: 8), NEEMHQAQRHHVQW (SEQ ID NO: 9), or A peptide comprising the amino acid sequence ALEPWGYKQVIKMAPN (SEQ ID NO: 10). 。

9. 9. The method of claim 8, wherein the cargo is a protein, a peptide, a nucleic acid, an antibody, or a chemical compound. The peptides listed.

10. 10. The peptide of claim 9, wherein the compound is an imaging agent.

11. The peptide of claim 9 , wherein the cargo is a therapeutic agent.

12. 12. The method of claim 8, wherein the peptide further comprises a second MTS peptide. The peptide described.

13. 13. The method of claim 12, wherein the second MTS peptide is the same as the first MTS peptide. The peptide described.

14. A composition comprising a peptide, wherein the peptide is a first peptide conjugated to a cargo. The first MTS peptide comprises: DAYKLQTSLDWQMWNP (SEQ ID NO: 1) DGYKLQTSLDWQMWNP (SEQ ID NO: 2), NQEYQHHKIKVRPSHQ (SEQ ID NO: 3), FPSWTSKNQQWTNQRQ (SEQ ID NO: 4), AHMSQKRLPHQVHQHQ (SEQ ID NO: 5), AGNKYEYTMHQKHNK (SEQ ID NO: 6), SKETYSMNAQRQHER (SEQ ID NO: 7), HRYDADRHHSFTPQYH (SEQ ID NO: 8), NEEMHQAQRHHVQW (SEQ ID NO: 9), or A composition comprising the amino acid sequence ALEPWGYKQVIKMAPN (SEQ ID NO: 10).

15. 15. The method of claim 14, wherein the cargo is a protein, a peptide, a nucleic acid, an antibody, or a chemical compound. The composition described.

16. 16. The composition of claim 15, wherein the compound is an imaging agent.

17. The composition of claim 154, wherein the cargo is a therapeutic agent.

18. 18. The composition of claim 14, further comprising a pharmaceutically acceptable carrier. The composition described in

19. The composition according to any one of claims 14 to 18, wherein the MTS has an N-terminal protecting group. thing.

20. 20. The composition of claim 19, wherein the N-terminal protecting group is an acetyl group.

21. the polypeptide comprises a linker between the first MTS peptide and the cargo. The composition according to any one of claims 14 to 20.

22. 22. The method of claim 21, wherein the linker is a polyethylene glycol (PEG) linker. The composition described above.

23. 23. Any one of claims 14 to 22, wherein the peptide further comprises a second MTS peptide. The composition described in

24. 24. The method of claim 23, wherein the second MTS peptide is the same as the first MTS peptide. The composition described.

25. A method for transporting cargo to the CNS of a subject, comprising administering to the subject a composition according to any one of claims 14 to 24. administering one or more of the compounds conjugated to a cargo to a subject in need thereof. wherein the lysed peptide enters the CNS.

26. 26. The method of claim 25, wherein the cargo-conjugated peptide enters the choroid plexus. method.

27. 27. The method of claim 25 or 26, wherein the polypeptide enters the CSF.

28. 28. The method of claim 27, wherein the CSF transports the cargo through the CNS.

29. 29. Any one of claims 25 to 28, wherein the cargo retains functional activity in the CNS. The method described in paragraph .

30. The method of any one of claims 25 to 29, wherein the administering is intravenous administration.

31. A method of treating a CNS disorder or injury in a subject, comprising administering to a subject a compound according to claims 1 to 24. administering one or more of the peptides or compositions to a subject in need thereof, The method, wherein the cargo is a therapeutic agent for a CNS disorder or injury.

32. The CNS disorder or injury is Parkinson's disease, Alzheimer's disease, glioblastoma, amyotrophic lateral sclerosis, or 32. The method of claim 31, wherein the condition is spondylosis, multiple sclerosis, or traumatic brain injury.

33. 33. The method of claim 31 or 32, wherein the CNS disorder treatment is an antibody or gene therapy. method.

34. A method for imaging the CNS of a subject, comprising administering to the subject a peptide or composition according to claims 1 to 24. to a subject in need thereof, wherein the cargo is an imaging agent That's the method.

Images