Molecular transport systems to the central nervous system

Abstract

This document discloses MTS peptides or targeting peptides. MTS peptides comprising the amino acid sequence of any one of SEQ ID NO: 1-12 are disclosed. Compositions comprising peptides, wherein the peptides comprise a first MTS peptide conjugated to a cargo, are disclosed. Methods of using MTS peptides are also disclosed. For example, a method of transporting cargo to a CNS is disclosed, the method comprising administering one or more of the disclosed compositions to a subject in need, wherein the cargo-conjugated peptide enters the CNS.

Description

[0001] Cross-referencing related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 991,465, filed March 18, 2020, which is incorporated herein by reference in its entirety.

[0003] References to sequence lists

[0004] The sequence list, created on March 16, 2021 and of size 3,389 bytes, submitted on March 18, 2021, is hereby incorporated by reference in accordance with 37C.FR§1.52(e)(5). Background Technology

[0005] Access to the central nervous system (CNS) is a bottleneck in the development of neurotherapies. This is due to the blood-brain barrier (BBB), a group of specialized and highly selective cellular barriers that protect the CNS. While necessary under normal physiological conditions, the BBB prevents many chemical entities, such as neurotherapeutic agents, from entering the brain. As a result, less than 5% of small molecule drugs pass through the BBB. Furthermore, newer biological therapies, such as antibodies and gene therapies, are essentially excluded from the CNS due to the BBB. Despite the discovery of the BBB over 100 years ago, a general solution for delivery to the CNS has not yet been developed. For this reason, many CNS conditions lack treatment options.

[0006] Currently, several other methods have been employed for delivery to CNS. One method is the use of BBB-permeable compounds. The problem with using BBB-permeable compounds is that they tend to have poor biodistribution properties and off-target effects (because they are often highly lipophilic). BBB-permeable compounds are also rare, as less than 5% of small molecules penetrate the BBB. BBB permeators also have limited indications.

[0007] Another method of entering the CNS is through direct injection into the spinal cord or brain. This method is invasive and carries the risk of structural damage to surrounding tissues and an increased risk of infection.

[0008] Disrupting the BBB is another method used. This method allows for the large-scale transport of compounds, cells, and pathogens into the CNS. This method can lead to structural damage and neuronal dysfunction.

[0009] Intranasal delivery has also been used. This method is limited to lipophilic small molecule drugs. Poor distribution via the CNS has been shown with intranasal delivery. Furthermore, intranasal delivery exhibits variable absorption between the dose and the patient.

[0010] Receptor-mediated delivery (Trojan horse) methods were also employed. This approach lacks versatility because it is typically effective only for a single shipment. Receptors can be expressed in multiple tissues, leading to poor CNS uptake and toxicity. Furthermore, most receptor-mediated deliveries lack cell specificity once they cross the BBB, and distribution throughout the CNS is inconsistent.

[0011] There are no known choroid plexus transporters or alternative methods for transporting cargo across the blood-brain barrier, thus presenting a unique development opportunity to influence the transport of drugs and macromolecules into the CNS. Therefore, this article discloses compositions and methods for targeting the CNS. Summary of the Invention

[0012] This paper describes a selection platform that utilizes choroid plexus biology to identify molecular transport system (MTS) peptides that can carry a variety of functional cargoes into the cerebrospinal fluid, whereby the cargoes can be distributed to cells of the CNS. This paper discloses MTS peptides for delivering cargoes (e.g., bioactive cargoes) from the CNS to the brain.

[0013] This document discloses MTS peptides or targeting peptides. Specifically, it discloses MTS peptides comprising the amino acid sequences of any one of SEQ ID NO: 1-12.

[0014] Peptides, including MTS peptides conjugated to cargo, are also disclosed.

[0015] Disclosed are compositions comprising peptides, wherein the peptides comprise a first MTS peptide conjugated to a cargo. Disclosed are compositions comprising peptides, wherein the peptides comprise a first MTS peptide conjugated to a cargo, wherein the first MTS peptide comprises the amino acid sequence of any one of SEQ ID NO: 1-12.

[0016] A method for transferring cargo to a CNS is disclosed, the method comprising administering one or more of the disclosed compositions to a subject in need, wherein a peptide conjugated to the cargo enters the CNS. In some aspects, the peptide bound to the cargo enters the choroid plexus.

[0017] A method for treating CNS conditions or injuries is disclosed, the method comprising administering one or more of the disclosed peptides or compositions to a subject in need, wherein the goods are therapeutic agents for CNS conditions or injuries.

[0018] A method for imaging the CNS is disclosed, the method comprising administering one or more of the disclosed peptides or compositions to a subject in need, wherein the cargo is an imaging agent.

[0019] Additional advantages of the disclosed methods and compositions will be set forth in part in the description which follows, and in part will be learned from the description, or may be acquired by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing general description and the following detailed description are exemplary and illustrative only, and are not intended to limit the claimed invention. Attached Figure Description

[0020] The accompanying drawings, which are incorporated in and form part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions.

[0021] Figure 1 This diagram illustrates the brain with the vascular endothelial barrier (commonly known as the blood-brain barrier (BBB)) and the choroid plexus. CSF = cerebrospinal fluid; CCSFB = choroidal cerebrospinal fluid barrier.

[0022] Figure 2 This is a schematic diagram illustrating the key features of the choroid plexus as a transport point to the CNS. The structural features of the fenestrated capillaries and the transcytosis activity of the choroid plexus epithelium are the basis for the functional differences between the choroid plexus and the BBB.

[0023] Figure 3 An unbiased selection experimental design for identifying MTS that mediate functional transport activities is shown.

[0024] Figure 4 An experimental design for confirming in vivo delivery of MTS to the CNS is shown.

[0025] Figure 5 It demonstrates how to develop an MTS system.

[0026] Figure 6 Tissue staining of CPEC4 phage clones in the choroid plexus and chambers is shown. The CPEC4 phage clone is a phage clone expressing the CPEC4 peptide (SEQ ID NO:2).

[0027] Figure 7 Tissue staining of the CPEC4 phage clone outside the ventricular system is shown.

[0028] Figure 8 This demonstrates that peptide engineering improves solubility and stability while maintaining transport capacity.

[0029] Figure 9 This study demonstrates the characteristics of maintaining the CNS barrier using a transwell model of primary human choroid plexus epithelial cells.

[0030] Figure 10 Unbiased selection of MTS for identifying mediating functional transport activities is shown.

[0031] Figure 11 The transwell selection of human primary choroid plexus epithelial cells is shown.

[0032] Figure 12 This demonstrates the validation of CPEC4 phage clone transport via human choroid plexus cells in an in vitro system.

[0033] Figure 13 The dimer CPEC4 peptide is shown.

[0034] Figure 14 This demonstrates the validation of CPEC4 phage clone transport via human choroid plexus cells in an in vitro system.

[0035] Figure 15 The transport of the CPEC4 phage clone and MTS_CPEC4_V1 via rat choroid plexus cells in an in vitro system is shown. The CPEC4 phage clone is a phage displaying the CPEC4 peptide (SEQ ID NO:2). MTS_CPEC4_V1 is a synthetic peptide of SEQ ID NO:2.

[0036] Figure 16 This demonstrates the confirmation of in vivo delivery systems in the CNS.

[0037] Figure 17 The study showed that the CPEC4 phage clone preferentially accumulated in rat CSF compared to the non-targeted empty phage clone.

[0038] Figure 18 The accumulation of MTS-CPEC4_V2 in rat CSF is shown. MTS_CPEC4_V2 comprises an MTS peptide having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO:1).

[0039] Figure 19 The differences in MTS_CPEC4 before and after stabilization are shown. MTS_CPEC4 comprises an MTS peptide with the sequence DGYKLQTSLDWQMWNP (SEQ ID NO:2).

[0040] Figure 20The synthesis of MTS_CPEC4_V2 using an isoacyl Thr-Ser dipeptide is illustrated. MTS_CPEC4_V2 comprises an MTS peptide having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO:1).

[0041] Figure 21 The percentage transport using the MTS_CPEC4_V2 dimer is shown. The MTS_CPEC4_V1 dimer comprises two MTS peptides, each having the sequence DGYKLQTSLDWQMWNP (SEQ ID NO:2); the MTS_CPEC4_V2 monomer comprises an MTS having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO:1); the MTS_CPEC4_V2 dimer comprises two MTS peptides, each having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO:1).

[0042] Figure 22 This demonstrates that MTS_CPEC4_V2 is stable in serum. MTS_CPEC4_V2 comprises an MTS peptide having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO:1).

[0043] Figure 23 The in vivo use of MTS_CPEC4_V2 is illustrated. MTS_CPEC4_V2 comprises an MTS peptide having the sequence DAYKLQTSLDWQMWNP (SEQ ID NO:1).

[0044] Figure 24 This is an experimental design for the in vitro and in vivo selection of lead MTS peptides.

[0045] Figure 25 The MTS_Z310-2 and MTS_Z310-5 peptides are shown to transport protein cargo across rat choroid plexus epithelial cells. MTS_Z310-2 comprises an MTS peptide having the sequence FPSWTSKNQQWTNQRQ (SEQ ID NO:4); MTS_Z310-5 comprises an MTS peptide having the sequence SKITYSMNAQRQHERS (SEQ ID NO:7).

[0046] Figure 26 The MTS_Z310-5 dimer was shown to enter the CSF and be observed in the ventricular system. The MTS_Z310-5 dimer comprises two MTS peptides, each having the sequence SKITYSMNAQRQHERS (SEQ ID NO:7).

[0047] Figure 27The MTS_Z310-2 dimer peptide was shown to enter the CSF and be observed in the ventricular system. MTS_Z310-2 comprises two MTS peptides, each having the sequence FPSWTSKNQQWTNQRQ (SEQ ID NO:4).

[0048] Figure 28 This demonstrates how dimer MTS can be combined together or combined with cargo.

[0049] Figure 29 An example of an MTS chimeric molecule is shown. Detailed Implementation

[0050] The disclosed methods and compositions can be more readily understood by referring to the following detailed description of specific embodiments and examples contained therein, as well as the accompanying drawings and their preceding and subsequent descriptions.

[0051] It should be understood that, unless otherwise stated, the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques, or specific reagents, and therefore may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0052] Materials, compositions, and components that can be used in conjunction with the disclosed methods and compositions, or as products of the disclosed methods and compositions, are disclosed herein. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, although specific references to every different individual and collective combination and arrangement of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a peptide is disclosed and discussed, and numerous modifications that can be made to multiple molecules comprising said peptide are discussed, then every combination and arrangement of said peptides and possible modifications are specifically considered unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and an example of the combination molecule AD is disclosed, then each is contemplated individually and collectively, even if each is not individually listed. Thus, in this example, each combination of AE, AF, BD, BE, BF, CD, CE, and CF is specifically contemplated and should be considered as disclosed from the disclosures of A, B, and C; D, E, and F; and the example combination AD. Similarly, any subset or combination of these combinations is specifically contemplated and disclosed. Thus, for example, subgroups of AE, BF, and CE are specifically contemplated and should be considered as disclosed in the disclosures of A, B, and C; D, E, and F; and the example combination AD. This concept applies to all aspects of this application, including but not limited to steps in methods of preparing and using the disclosed compositions. Therefore, if various additional steps are available, it should be understood that these additional steps can each be performed using any particular embodiment or combination of embodiments of the disclosed methods, and each such combination is particularly contemplated and should be considered as disclosed.

[0053] A. Definition

[0054] It should be understood that the disclosed methods and compositions are not limited to the specific methods, schemes, and reagents described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims.

[0055] It should be noted that, as used herein and in the appended claims, the singular forms “a / an” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, references to “peptide” include multiple such peptides, and references to “composition” refer to one or more compositions known to those skilled in the art, as well as their equivalents.

[0056] As used herein, “treatment” means administering the compositions of the present invention to a subject suffering from a disease or condition, such as a human or other mammal (e.g., an animal model), in order to prevent or delay the worsening of the effects of the disease or condition or to partially or completely reverse the effects of the disease or condition. In some aspects, a disease or condition may be a CNS-related disease or condition or a CNS symptom or lesion. For the purpose of reducing the risk of developing a pathology associated with a disease, condition, and / or condition, treatment may be administered to subjects who do not exhibit signs of a disease, condition, and / or condition and / or subjects who exhibit only early signs of a disease, condition, and / or condition. In some embodiments, treatment comprises delivering one or more of the disclosed compositions to a subject.

[0057] As used in this article, “prevention” means minimizing the chances of a subject with an increased susceptibility to developing a disease, condition, or symptom.

[0058] As used herein, the term "subject" refers to the target of the application, such as a human. Therefore, the subject of the disclosed methods can be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). On the one hand, the subject is a mammal. On the other hand, the subject is a human. The terms do not indicate a specific age or sex. Therefore, it is intended to encompass adult subjects, child subjects, adolescent subjects, newborn subjects, and fetuses, whether male or female.

[0059] As used herein, the term "patient" refers to a subject suffering from a disease or condition. The term "patient" includes both human and animal subjects. In some aspects of the disclosed methods, the "patient" has been diagnosed as requiring treatment prior to the administration of the procedure. In some aspects, the terms "patient" and "subject" are used interchangeably.

[0060] As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, characters, or words representing amino acid residues. The amino acid abbreviations used herein are the standard single-letter codes for amino acids and are 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.

[0061] As used herein, "polypeptide" refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. Polypeptides consist of a continuous sequence of amino acids. The term "polypeptide" encompasses both naturally occurring and synthetic molecules.

[0062] Furthermore, as used herein, the term "peptide" refers to amino acids linked together by peptide bonds or modified peptide bonds, such as isosteric peptides, and may contain modified amino acids in addition to the 20 amino acids encoded by genes. Peptides can be modified by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Modifications can occur anywhere in a peptide, including the peptide backbone, amino acid side chains, and amino or carboxyl terminals. The same type of modification can be present at several sites on a given peptide in the same or different amounts. Similarly, a given peptide can have many types of modifications. Modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent crosslinking or cyclization, covalent linkage of flavin, covalent linkage of heme moieties, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine ​​or pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolylation, oxidation, pergylation, proteolytic processing, phosphorylation, isopreneation, racemization, selenylation, sulfation, and transfer-RNA-mediated addition of amino acids to proteins, such as arginylation. (See Proteins—Structure and Molecular Properties, 2nd ed., TECreighton, WH Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, edited by BC Johnson, Academic Press, New York, pp. 1-12 (1983).)

[0063] As used herein, the phrase "nucleic acid sequence" refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA, RNA, or a DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, capable of hybridizing with complementary nucleic acids via Watson-Crick base-pairing. The nucleic acid sequences of this invention may also contain nucleotide analogs (e.g., BrdU) and nonphosphodiester nucleoside bonds (e.g., peptide nucleic acid (PNA) or thiodiester bonds). Specifically, the nucleic acid sequence may include, but is not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0064] As used herein, an “effective amount” of a composition means an amount of the composition sufficient to provide the desired effect. The exact amount required will vary from subject to subject, depending on the subject’s species, age and general condition, the severity of the disease being treated (or the underlying genetic defect), the specific compound used, its administration method, etc. Therefore, it is not possible to specify an exact “effective amount.” However, those skilled in the art can determine an appropriate “effective amount” using only routine experiments.

[0065] As used herein, “selective binding” means that a nucleic acid sequence (e.g., cargo) or MTS recognizes and physically interacts with its target (e.g., a specific cell type) without significantly recognizing and interacting with other targets.

[0066] The term “percentage of homology (%)” is used interchangeably with the term “percentage of identity (%)” herein and refers to the level of nucleic acid or amino acid sequence identity when aligned with a wild-type sequence or a sequence of interest using a sequence alignment program. For example, as used herein, 80% homology means something that is identical to 80% of the sequence identity determined by a defined algorithm, and therefore, homologs of a given sequence have greater than 80% sequence identity over the length of the given sequence. Exemplary levels of sequence identity as described herein include, but are not limited to, 80%, 85%, 90%, 95%, 98% or greater sequence identity with a given sequence, such as any MTS sequence, such as an MTS sequence. Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, BLAST program suites publicly available on the Internet, such as BLASTN, BLASTX and TBLASTX, BLASTP and TBLASTN. See also, Altschul et al., 1990 and Altschul et al., 1997. When evaluating a given nucleic acid sequence relative to nucleic acid sequences in GenBank DNA Sequence and other public databases, the BLASTN program is typically used for sequence searching. For searching for nucleic acid sequences that have been translated across all reading frames against amino acid sequences in GenBank protein Sequence and other public databases, the BLASTX program is preferred. Both BLASTN and BLASTX are run with the default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0, and use a BLOSUM-62 matrix. (See, for example, Altschul, SF et al., Nucleic Acids Res. 25:3389-3402, 1997). To determine the “% of identity” between two or more sequences, a preferred alignment of selected sequences is performed using, for example, the CLUSTAL-W program in Mac Vector version 13.0.7, which operates with default parameters including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0067] Substitution, deletion, insertion, or any combination thereof can be used to obtain the final derivative, variant, or analogue. Typically, these changes are made on a few nucleotides to minimize the alteration of the molecule. However, larger changes can be tolerated in certain cases.

[0068] Typically, the nucleotide identity between variant sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Therefore, a "variant sequence" can be a sequence that has a specified identity with the parent or reference sequence of the present invention (e.g., a wild-type sequence) and shares a biological function, which includes, but is not limited to, 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 specificity and / or activity of the parent sequence. For example, a "variant sequence" can be a sequence that contains one, two, three, or four nucleotide base changes compared to the parent or reference sequence of the present invention and shares or improves the biological function, specificity, and / or activity of the parent sequence. Therefore, a "variant sequence" can be a sequence that has designated identity with the parental sequence of the present invention and shares a biological function, which includes, but is not limited to, 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 specificity and / or activity of the parental sequence. The variant sequence may also share 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 specificity and / or activity of the reference sequence (e.g., the MTS sequence).

[0069] "Optional" or "optionally" means that the event, environment, or material described below may or may not occur or exist, and the description includes situations in which the event, environment, or material occurs or exists, as well as situations in which it does not occur or does not exist.

[0070] A range herein may be expressed as from “about” one particular value and / or to “about” another particular value. When expressing such a range, unless the context explicitly states otherwise, a range from one particular value and / or to another particular value is also specifically contemplated and considered disclosed. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value forms another specifically contemplated embodiment, which, unless the context explicitly states otherwise, should be considered disclosed. It will be further understood that, unless the context explicitly states otherwise, the endpoints of each range are significant relative to and independent of the other endpoint. Finally, it should be understood that, unless the context explicitly states otherwise, all individual values ​​and subranges of values ​​contained within the explicitly disclosed range are also specifically contemplated and should be considered disclosed. The foregoing applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular case.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions pertain. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the methods and compositions of the invention, particularly useful methods, apparatus, and materials are as described. Publications cited herein, and the materials to which they pertain, are specifically incorporated herein by reference. Nothing herein should be construed as an admission that the invention is not entitled to precedence by any prior invention. No cited document is acknowledged as prior art. The discussion of the references illustrates the author's claims, and the applicant reserves the right to challenge the accuracy and relevance of cited documents. It should be clearly understood that although numerous publications are cited herein, such citations do not constitute an admission that any of these documents constitutes part of common general knowledge in the art.

[0072] Throughout the description and claims of this specification, the word "comprise" and variations thereof, such as "comprising" and "comprises," mean "including but not limited to" and are not intended to exclude, for example, other additives, components, integers, or steps. In particular, in a method described as comprising one or more steps or operations, each step is specifically envisioned to include the listed contents (unless the step contains restrictive terms such as "consisting of"), meaning that each step is not intended to exclude, for example, other additives, components, integers, or steps not listed in the step description.

[0073] B peptides

[0074] Peptides that can provide molecular transport or target a specific location or site can be called MTS peptides. MTS peptides are amino acid sequences known as molecular transport systems because they target a specific location or site, thus enabling them to transport cargo to that location or site. In some respects, the MTS peptides disclosed herein target the CNS (Central Nerve System).

[0075] This document discloses MTS peptides or targeting peptides. MTS peptides are disclosed that comprise amino acid sequences of any of the sequences shown in SEQ ID NO:1-10 as shown in Table 1. In some aspects, MTS peptides consist of amino acid sequences responsible for molecular transport or targeting of a specific site or location. For example, in some aspects, the disclosed MTS peptides consist of amino acid sequences of any of the sequences shown in SEQ ID NO:1-10 as shown in Table 1.

[0076] Table 1. Example MTS peptides

[0077]

[0078]

[0079] In some aspects, the one or more MTS peptides have sequence identity with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the sequences shown in SEQ ID NO:1-10. In some aspects, the one or more MTS peptides have sequence identity with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the MTS peptides disclosed herein. In some aspects, the one or more MTS peptides have 100% identity in the active portion of the peptide, which is the portion that retains its ability to cross from the bloodstream into the CSF. Therefore, in some aspects, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with any MTS peptide appears outside the active portion.

[0080] In some respects, MTS peptides can be modified. Modifications of MTS peptides include optimizing or stabilizing peptides.

[0081] In some respects, MTS peptides can be stabilized so that they remain intact (e.g., do not degrade) during synthesis and / or storage. In some respects, the MTS peptide comprising the sequence DGYKLQTSLDWQMWNP (SEQ ID NO:2) can be stabilized by changing glycine to alanine at the second amino acid position in DAYKLQTSLDWQMWNP (SEQ ID NO:1), and can be further optimized with an N-terminal acetyl protecting group. In some respects, the aspartic and glycine dipeptide (DG of SEQ ID NO:2) can form a cyclic intermediate (see [link to relevant documentation]). Figure 19 The cyclic intermediate can be opened to form the original MTS peptide or non-natural amino acids, thus producing a less stable MTS peptide.

[0082] In several respects, MTS peptides can be optimized. Optimized peptides can be obtained by modifying the individual parent peptide sequences. These modifications can be used to identify the essential amino acids required within the parent sequence to cross from the blood into the CSF. These modifications can be obtained through a combination of alanine scanning and truncation of the N-terminal and C-terminal regions of the parent peptide. PEG11 can provide protection for the C-terminus of the MTS peptide, provide a spacer between the peptide and the cargo molecule linked via a cysteine ​​residue at the C-terminus, and enhance the solubility of the MTS peptide. Modification at the N-terminus by acetylation (CH3CO-) and / or d-amino acids such as d(Leu) can prevent degradation by peptidases in the blood. There is no single optimized peptide of uniform length that can be applied to all MTS peptides, and all variations can be tested to confirm their effects on peptide absorption and stability.

[0083] In some aspects, the MTS peptides disclosed herein may have an N-terminal protecting group. In some aspects, the N-terminal protecting group may be any substance that prevents proteases from cleaving amino acids from the N-terminus. In some aspects, the MTS peptides disclosed herein may be modified by acetylation at the N-terminus. In some aspects, the N-terminal protecting group is an acetyl group. Therefore, in some aspects, the MTS peptides disclosed herein may be acetylated. In some aspects, the N-terminal protecting group may be, but is not limited to, polyethylene glycol, formyl, CH3-(CH)n-CO, fluorophore, fatty acid, alkylamine, sulfonamide, or carbamate. In some aspects, the MTS peptides disclosed herein may be chemically conjugated to goods such as nucleic acid sequences. In some aspects, the chemical conjugate may be polyethylene glycol (PEG). Therefore, in some aspects, the MTS peptides disclosed herein may be polyethylene glycolated. In some aspects, the number of PEG units may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more. In some aspects, the number of PEG units can be long enough to separate one or more MTS peptides from the cargo, thereby preventing any steric interference between the one or more MTS peptides and the cargo. Therefore, in some aspects, the MTS peptides disclosed herein may further include a linker. For example, linkers and chemical conjugates can be used interchangeably. In some aspects, the linker is located at the C-terminus of the MTS peptide. For example, compositions comprising a chemical conjugate or linker are disclosed herein, wherein the chemical conjugate or linker is PEG and the PEG comprises eleven PEG units. In one aspect, the MTS peptides disclosed herein comprise one or more sequences shown in SEQ ID NO:1-10, wherein SEQ ID NO:1-10 can be acetylated at the N-terminus and can be chemically conjugated to PEG; and cargo such as nucleic acid sequences can also be covalently linked to PEG. In some aspects, the N-terminal protecting group can be an artificial amino acid such as a D-amino acid.

[0084] On one hand, the MTS peptide disclosed herein can be truncated. In some aspects, the MTS peptide disclosed herein is truncated to eliminate all amino acids except the active portion of the MTS peptide. In some aspects, the active portion of the MTS peptide can be used in the disclosed compositions and methods. In some aspects, the active portion can be determined using techniques well known in the art, such as alanine scanning or truncation studies. The active portion of the MTS peptide is the portion that retains its ability to cross from the bloodstream into the CSF. For example, SEQ ID NO:7 can be truncated at the N-terminus by up to four amino acids. In some aspects, SEQ ID NO:7 can be truncated at the C-terminus by up to eight amino acids. In some aspects, the amino acid sequence YSMNAQRQHERS (SEQ ID NO:11) is the active portion of SEQ ID NO:7. In some aspects, the amino acid sequence YSMN (SEQ ID NO:12) is the active portion of SEQ ID NO:7. Therefore, in some aspects, SEQ ID NO:12 can be used as an MTS peptide.

[0085] In some respects, stable variants of the MTS peptides disclosed herein are disclosed.

[0086] Peptides are also disclosed, including MTS peptides conjugated to cargo. In some aspects, two or more MTS peptides may be conjugated to cargo.

[0087] A peptide is disclosed, the peptide comprising a first MTS peptide conjugated to cargo, wherein the first MTS peptide comprises an amino acid sequence of any sequence shown in SEQ ID NO: 1-12.

[0088] In some respects, the cargo molecule can be, but is not limited to, nucleic acid sequences, proteins, antibodies, peptides, nanoparticles, dyes, compounds, or small molecules. As described herein, the cargo is a nucleic acid sequence. In some respects, the cargo can be an imaging agent, a radionuclide, or a detectable marker.

[0089] Peptides are disclosed that comprise a first MTS peptide conjugated to cargo and further comprise a second MTS peptide. For example, peptides are disclosed that comprise a first MTS peptide conjugated to cargo and further comprise a second MTS peptide, wherein the first MTS peptide comprises an amino acid sequence of any sequence shown in SEQ ID NO:1-12.

[0090] In some respects, the second MTS peptide is the same as the first MTS peptide. In other respects, the second MTS peptide differs from the first MTS peptide.

[0091] The connection between the MTS and the cargo can be achieved through maleimide chemistry, click chemistry, amide chemistry, and hydrazone chemistry. Cuttable or non-cuttable connectors can be used to connect the cargo.

[0092] MTS dimers can be synthesized using linear peptide chemistry, such as FMOC (maleimide-free). The cargo can then be linked to the dimer via maleimide chemistry, click chemistry, amide chemistry, and hydrazone chemistry.

[0093] Examples of how to prepare conjugates can be found in Figure 28 and Figure 29 middle.

[0094] C. Composition

[0095] Compositions comprising one or more of the disclosed peptides are disclosed. In some aspects, compositions comprising one or more MTS peptides disclosed herein are disclosed. Compositions comprising peptides, wherein the peptides comprise a first MTS peptide conjugated to a cargo, are also disclosed.

[0096] For example, compositions comprising peptides are disclosed, wherein the peptides comprise a first MTS peptide conjugated to cargo, wherein the first MTS peptide comprises an amino acid sequence of any sequence shown in SEQ ID NO:1-12.

[0097] 1. Pharmaceutical composition

[0098] In some aspects, the disclosed compositions may be pharmaceutical compositions. For example, in some aspects, pharmaceutical compositions are disclosed comprising a composition including a nucleic acid sequence conjugated to one or more MTS peptides and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material or carrier, as is well known to those skilled in the art, that will be selected to minimize any degradation of the active ingredient and any adverse side effects on the subject. Examples of carriers include dimyristoylphosphatidyl (DMPC), phosphate-buffered saline, or multivesicular liposomes. For example, PG:PC:cholesterol:peptide or PC:peptide can be used as a carrier in this invention. Other suitable pharmaceutically acceptable carriers and formulations thereof are described in Remington: The Science and Practice of Pharmacy (19th edition), edited by ARGennaro, Mack Publishing Company, Easton, PA, 1995. Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Other examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextran solution. The pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5. Other carriers comprise sustained-release formulations, such as a semi-permeable matrix containing a solid hydrophobic polymer of the composition, said matrix being in the form of a molded article, such as a membrane, a stent (implanted into a blood vessel during angioplasty), liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be preferred, depending, for example, on the route of administration and the concentration of the composition administered. These most typically will be standard carriers used for administering drugs to humans, comprising solutions such as sterile water, saline, and buffer solutions at physiological pH.

[0099] The pharmaceutical composition may also contain carriers, thickeners, diluents, buffers, preservatives, etc., as long as they do not impair the intended activity of the polypeptide, peptide, or conjugate of the present invention. The pharmaceutical composition may also contain one or more active ingredients (other than those in the compositions of the present invention), such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc.

[0100] The pharmaceutical compositions disclosed herein can be prepared for oral or parenteral administration. Preparation of pharmaceutical compositions for parenteral administration includes those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), or transdermal (e.g., topical) administration. Nebulized inhalation can also be used to deliver fusion proteins. Therefore, compositions for parenteral administration can be prepared comprising a fusion protein dissolved or suspended in an acceptable carrier, including, but not limited to, aqueous carriers such as water, buffered water, saline, buffered saline (e.g., PBS), etc. One or more of the included excipients can help approximate physiological conditions, such as pH adjusters and buffers, tension modifiers, wetting agents, detergents, etc. In the case where the composition contains a solid component (as the composition is intended for oral administration), one or more of the excipients can act as a binder or filler (e.g., for the formulation of tablets, capsules, etc.). When the composition is formulated for application to the skin or mucous membrane surface, one or more of the excipients may be solvents or emulsifiers used for formulating creams, ointments, etc.

[0101] The pharmaceutical composition may be sterile and sterilized by conventional sterilization techniques or aseptic filtration. Aqueous solutions may be packaged for use as is or lyophilized; lyophilized formulations covered by this disclosure may be combined with a sterile aqueous carrier prior to administration. The pH of the pharmaceutical composition will generally be between 3 and 11 (e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7 and 8). The resulting solid form composition may be packaged in multiple single-dose units, each containing a fixed amount of one or more of the aforementioned pharmaceutical agents, as in a sealed package of tablets or capsules. The solid form composition may also be packaged in containers for flexible dosages, such as in squeeze tubes designed for topical application of creams or ointments.

[0102] The pharmaceutical compositions described above can be formulated to contain a therapeutically effective amount of the compositions disclosed herein. In some respects, therapeutic administration encompasses prophylactic application. Based on genetic testing and other prognostic methods, a physician may choose prophylactic administration, in consultation with the patient and physician, where the patient has a clinically established predisposition or increased susceptibility (in some cases, a significantly increased susceptibility) to one or more autoimmune diseases, or where the patient has a clinically established predisposition or increased susceptibility (in some cases, a significantly increased susceptibility) to cancer.

[0103] The pharmaceutical compositions described herein may be administered to subjects (e.g., human subjects or human patients) in amounts sufficient to delay, reduce, or preferably prevent the onset of clinical disease. Thus, in some aspects, the subject is a human subject. In therapeutic applications, the composition is administered to subjects (e.g., human subjects) who already have or have been diagnosed with a CNS disease or condition in an amount sufficient to at least partially improve signs or symptoms or inhibit (and preferably prevent) the progression of the condition, its complications, and consequences. The amount sufficient to achieve this is defined as a “therapeuticly effective amount.” A therapeutically effective amount of the pharmaceutical composition may be an amount that achieves a cure, but this result is only one of several possible outcomes. As mentioned above, a therapeutically effective amount includes an amount that provides treatment, wherein the onset or progression of cancer is delayed, inhibited, or prevented, or the CNS disease or condition or its symptoms are improved. One or more of the symptoms may be less severe. Individuals who have been treated may experience accelerated recovery.

[0104] The total effective amount of the conjugates in the pharmaceutical compositions disclosed herein can be administered to mammals as a single dose, as a bolus, or by infusion over a relatively short period of time, or can be administered using a fractionated treatment regimen, wherein multiple doses are administered over a longer period of time (e.g., every 4 to 6, 8 to 12, 14 to 16, or 18 to 24 hours, or every 2 to 4 days, 1 to 2 weeks, or monthly). Alternatively, continuous intravenous infusion sufficient to maintain therapeutically effective concentrations in the blood is also within the scope of this disclosure.

[0105] D. Method

[0106] Methods using one or more of the disclosed peptides (e.g., MTS peptides) or compositions are disclosed herein. Any of the peptides (e.g., MTS peptides) or compositions disclosed herein may be used in the methods disclosed herein. In some aspects of the disclosed methods, the MTS peptide conjugated with the cargo may cross from the bloodstream into the CNS.

[0107] 1. Methods of transshipping goods

[0108] A method for transporting cargo to the CNS is disclosed, the method comprising administering one or more of the disclosed compositions to a subject in need, wherein an MTS peptide conjugated with the cargo enters the CNS. In some aspects, the MTS peptide conjugated with the cargo enters the choroid plexus. In some aspects, the MTS peptide conjugated with the cargo enters the cerebrospinal fluid (CSF). In some aspects, the CSF transports the MTS peptide conjugated with the cargo throughout the CNS. In some aspects, the MTS peptide can be cleaved from the cargo, resulting in the CSF transporting the cargo throughout the CNS, separating from the MTS peptide.

[0109] In the disclosed method, once the goods are delivered to the CNS, they retain their functional activity within the CNS.

[0110] In some respects, administration is intravenous. In other respects, administration is intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal), or transdermal (e.g., local) administration. In some respects, administration may be by nebulization.

[0111] 2. Treatment methods

[0112] A method for treating CNS conditions or injuries is disclosed, the method comprising administering one or more of the disclosed peptides (e.g., MTS peptides) or compositions to a subject in need, wherein the goods are therapeutic agents for CNS conditions or injuries.

[0113] In some respects, CNS symptoms or lesions can be, but are not limited to, Parkinson's disease, Alzheimer's disease, glioblastoma and other cancers that have metastasized to the brain, amyotrophic lateral sclerosis, multiple sclerosis, and traumatic brain injury. Therefore, in some respects, therapeutic agents for CNS symptoms or lesions can be, but are not limited to, antibodies, gene therapies, compounds, nucleic acid sequences, or peptides (or proteins). Specific examples of therapeutic agents for CNS symptoms or lesions in some respects can be, but are not limited to, N-methyl-D-aspartate (NMDA) antagonists, chemotherapeutic agents, glutamate antagonists, or immunomodulators (e.g., immunosuppressants or immune activators).

[0114] 3. Imaging methods

[0115] A method for imaging the CNS is disclosed, the method comprising administering one or more of the disclosed peptides (e.g., MTS peptides) or compositions to a subject in need, wherein the cargo is an imaging agent. As used herein, imaging agent, imaging marker, or imaging portion (also referred to as detectable marker, detectable portion, or diagnostic portion) refers to an atom, molecule, or composition, wherein the presence of the atom, molecule, or composition can be measured directly or indirectly.

[0116] In some aspects, the imaging agent may be, but is not limited to, a fluorescent dye, a radioactive isotope, magnetic beads, metal beads, colloidal particles, near-infrared dyes, or an electron-dense reagent. Therefore, the detectable portion may be, but is not limited to, a fluorescent portion, a radioactive portion, or an electron-dense portion. In some aspects, the imaging agent comprises two portions, wherein portion one includes a disease-specific peptide, nucleic acid, or compound conjugated to portion two, and portion two includes a fluorescent dye, a radioactive isotope, magnetic beads, metal beads, colloidal particles, near-infrared dyes, or an electron-dense reagent. In some aspects, portion one of the imaging agent binds to a disease-specific indicator in the CNS, and portion two of the imaging agent allows for the detection or visualization of the imaging agent. In some aspects, the disease-specific peptide, nucleic acid, or compound may bind to a protein or nucleic acid in the CNS that indicates a specific disease, such as, but not limited to, Parkinson's disease, Alzheimer's disease, glioblastoma and other cancers that metastasize to the brain, amyotrophic lateral sclerosis, multiple sclerosis, and traumatic brain injury. The binding of disease-specific peptides, nucleic acids, or compounds to proteins or nucleic acids in the CNS that indicate a specific disease can be detected or visualized by part 2 of an imaging agent.

[0117] The disclosed method for imaging the CNS allows for monitoring of the CNS in the presence of disease or disease progression. For example, once the presence of disease or disease progression is detected, a second MTS peptide conjugated to a cargo can be administered to the subject, wherein the cargo is a therapeutic agent. In some aspects, if the first peptide comprises an MTS peptide conjugated to a cargo, wherein the cargo is an imaging agent for identifying the presence of glioblastoma in the CNS, then the second peptide may comprise an MTS peptide conjugated to a cargo, wherein the cargo is a glioblastoma-specific therapeutic agent.

[0118] A method for imaging the CNS is also disclosed, the method comprising administering a peptide, said peptide comprising one or more of the disclosed MTS peptides conjugated to a first cargo and a second cargo, wherein the first cargo is an imaging agent and the second cargo is a therapeutic agent for CNS symptoms or lesions. Thus, in some aspects, the CNS can be imaged, and if the presence or progression of disease is detected, the therapeutic agent for CNS symptoms or lesions can provide a therapeutic effect.

[0119] E. Reagent kit

[0120] The above-described materials, along with other materials, can be packaged together in any suitable combination as a kit for performing or assisting in the performance of the disclosed methods. A given kit is useful if its components are designed and suitable for use in the disclosed methods. For example, a kit comprising one or more of the disclosed peptides is disclosed.

[0121] Example

[0122] DiaCyt is a novel platform technology for identifying delivery agents that selectively transport a variety of therapeutic cargoes into the CNS without physically disrupting the BBB. A selective platform for identifying peptide-based molecular transport systems (MTS) has been developed: capable of entering the CNS without disrupting the barrier; delivering therapeutic agents containing biomolecules such as antibodies, proteins, and nucleic acids into the CNS without inactivating the therapeutic agent during transport; and releasing the MTS cargo and distributing the cargo throughout the CNS.

[0123] This concept is unique in two ways. First, selection is an unbiased phenotypic approach that allows for the selection of MTSs possessing the key characteristics described above. This identifies MTSs capable of crossing the CNS barrier and being released into the CNS. Furthermore, selection requires transporting functional phages. Thus, the identified MTSs carry biological material through the cell without degrading the cargo during the process.

[0124] The second unique feature is that, unlike the endothelial BBB, the choroid plexus is targeted. This is advantageous because it utilizes the physiological structure and biological function of the choroid plexus (see Appendix) and overcomes delivery barriers through the endothelial BBB, which is already the focus of the field. The capillaries of the choroid plexus are porous (more permeable) and lack astrocyte foot processes around the vessels, which allows material to pass through from the blood.

[0125] Figure 1 The BBB is a significant biological barrier. Administering drugs and biotherapeutic agents to CNS tissues via intercellular spaces is not an option. The peptides, compositions, and methods disclosed herein provide a new paradigm for drug development. The choroid plexus produces cerebrospinal fluid (CSF). Epithelial cells have tight junctions that form the blood-cerebrospinal fluid barrier (BCSFB). Unlike the BBB, capillaries are porous (spaces between endothelial cells), allowing immune cells, proteins, and even pathogens to escape. Choroid plexus epithelial cells are specialized cells that transport ions, peptide hormones, and proteins from the blood to the CSF. Tight junctions are absent in the ependyma, pia mater, and glial boundary membrane, allowing paracellular diffusion of CSF between the brain and the ependyma.

[0126] Figure 2A schematic diagram illustrating capillaries in the choroid plexus and how these capillaries transport cargo into the CNS is shown. Some structural advantages of targeting the choroid plexus include 1) the capillaries being porous (more permeable), allowing material to pass through the bloodstream and 2) the absence of astrocyte foot processes around the vessels. Some biological advantages of targeting the choroid plexus include its utilization of the natural function of choroid plexus epithelial cells in transporting molecules, peptides, proteins, and cells into the CNS. Another biological advantage of targeting the choroid plexus is its ability to transport cargo into the CSF, which then circulates throughout the CNS.

[0127] An experimental design for unbiased selection to identify MTS peptides mediating functional transport activities is shown in Figure 3 The identified MTS can be easily optimized chemically to produce an MTS suitable for delivering goods to CNS.

[0128] An experimental design for confirming in vivo delivery of MTS to CNS is shown in Figure 4 This design allows for the selection of MTSs that mediate functional transport across the cell barrier by protecting cargo from degradation.

[0129] Figure 5 This paper outlines how a system was developed to identify MTS peptides. This system identified an MTS phage clone capable of transcytosis in an in vitro model and entry into CSF ​​in an in vivo model. Data on the synthesis of MTS_CPEC4 (DGYKLQTSLDWQMWNP (SEQ ID NO:2)) demonstrate its in vitro and in vivo activity.

[0130] Figure 6 Tissue staining of choroid plexus and CPEC4 phage clones in chambers is shown, and Figure 7 Histological staining of the CPEC4 phage clone outside the ventricular system is shown. This indicates that delivery of MTS-cargo to the CNS will result in cargo translocation to other areas of the brain. The phage clone was injected intravenously via the tail vein of a rat. After a set time period for phage clone circulation, the animal underwent final perfusion and the brain was isolated. After fixation, the brain was sectioned and processed for immunohistochemistry. Phages were detected using a primary antibody against M13 phage. Blue staining is nuclear staining. Phage staining was observed in the ventricular and peripheral tissues, indicating phage clone translocation to the CSF.

[0131] This data established an in vitro model of the choroid plexus using tightly connected human primary choroid plexus epithelial cells found in replicated human choroid plexus. A rat model was also developed. The selection of phage clones that cross the choroid plexus epithelial barrier from the library was also communicated. The peptides were synthesized outside the phage environment, and it was demonstrated that the peptides retained their transport activity and could carry small molecules and proteins across the barrier. Phage clones were selectively transported into CSF, and data support similar transport of the MTS peptide.

[0132] Once an MTS peptide is identified, it can be optimized through several methods involving chemical modifications. Optimization can increase solubility, stability, brain distribution, biodistribution, cellular transport, or circulation time. The desired outcome with MTS is an injection dose present in at least 2.5% of the CNS, but not exceeding 5% of the CNS.

[0133] Current research screens and validates in in vitro transwells, provides targeting in animal models using multiple methods with fully validated delivery efficiency (real-time CSF analysis, ex vivo analysis of CSF, and accompanying histological, radiolabeling, and / or imaging), and assesses the impact of different cargoes on MTS delivery efficiency (peptides, proteins, and nucleic acids).

[0134] Figure 10 This is a schematic diagram of an experimental design using a monolayer of rat choroidal epithelium (Z310 cells) within a transwell bucket for unbiased selection to identify MTS peptides mediating functional transport activity. Z310 cells were exposed to a phage library on their basal side (the blood side mimicking the choroid plexus), where transported phages were present at the apex (CSF side, inside the bucket) after incubation. Following this incubation, phages were also recovered from cell lysates. The library used for this selection expressed a 16-mer peptide generated by randomly adding synthetic codons.

[0135] Figure 11 The abundance of the peptide sequence encoding MTS_CPEC4_V1 (SEQ ID NO:2) was summarized from sequences obtained from 96 individual phage clones submitted in rounds 4 and 5 for DNA sequencing by transwell and lysate samples. MTS_CPEC4_V1 was selected from a random 16-meric peptide library with an initial complexity of 2.6 × 10⁻⁶. 10 This peptide clone was the dominant sequence observed in the fourth and fifth rounds of selection. Figure 11The frequency of this specific sequence observed in the outputs of rounds 4 and 5 was summarized. As shown, this sequence was observed in both transwell transport outputs and cell lysates at the end of timed incubation. A total of 144 out of 365 CPEC4_V1 clones were observed in the outputs of rounds 4 and 5. The only other peptide observed in both rounds' transwell outputs and cell lysates was at a much lower frequency (6 / 365)...

[0136] Transwell selection of human primary choroid plexus epithelial cells has been completed and a lead peptide has been generated. Figure 12 The CPEC4 phage clone sequence DGYKLQTSLDWQMWNP (SEQ ID NO:2) is the lead peptide sequence in these studies. CPEC4 phage clones were enriched through multiple rounds of panning. The CPEC4 phage clones were selected from transwells indicating transport and release. The presence of CPEC4 phage clones in cell lysates indicates active transport through the cell rather than "around" the cell.

[0137] Figure 12 This study demonstrates that the CPEC4 peptide (SEQ ID NO:2) mediates the transport of macrophage particles across the choroid plexus epithelial cell layer. The phage clone remains viable during cellular transport.

[0138] Figure 13 This represents a dimer of the CPEC4 peptide. The CPEC4 peptide is synthesized as a dimer and has a biotin handle that allows for the attachment of fluorescently labeled proteins.

[0139] Figure 14 The transport of fluorescent molecules across the human vascular plexus plexus mediated by MTS_CPEC4_V1 (SEQ ID NO:2) in an in vitro model was demonstrated. 6% to 9% of the protein was transported across the cell layer. Tight junctions remained intact, as witnessed by the lack of dextran transport and maintenance of transepithelial resistance at the end of the experiment.

[0140] Figure 15This is an in vitro transwell assay used to determine the ability of MTS_CPEC4 phage clones or peptides to cross a choroid plexus barrier model derived from rats (Z310 cells). The amount of phage placed on one side of the barrier (“blood” side) and the amount that crosses the cell to the other side (CSF side) are measured. The table above shows data for phage clones. Empty phages do not have attached MTS peptides. The amount of phage in each side of the transwell is determined by bacterial titer. The table below shows data for synthetic peptides. Here, peptides are labeled with fluorophores so that peptides on either side of the barrier can be measured. A dextran-dye (such as Alexa Fluor 488) is used as a control molecule. This molecule should not cross the transwell and ensures the choroid plexus model is valid.

[0141] Figure 16 This diagram illustrates how the MTS delivery system targets the CNS. Following tail vein injection in mice, CSF can be removed from the brain via capillary puncture of the cerebellomedullary cistern, and the presence of bacteriophages can be confirmed.

[0142] Figure 17 The study showed that, compared to the control phage, CPEC4 phage clones preferentially accumulated in CSF, thus mediating CSF transport. The phage clones remained viable during transport to CSF.

[0143] Figure 18 The experimental design and final results of CSF accumulation in rats after tail vein injection are shown: MTS-CPEC4_V2 (SEQ ID NO:1).

[0144] Although MTS can be synthesized, it is difficult to synthesize high-quality monomeric peptides exceeding one milligram, and dimers are even more challenging. It has been determined that inserting “TS” into the middle of the MTS_CPEC4_V2 sequence using an isoacyl Thr-Ser dipeptide provides beneficial results. Figure 20 ).

[0145] like Figure 21 As shown, CPEC4_V1 (SEQ ID NO:2) and CPEC_V2 (SEQ ID NO:1) efficiently transport chemicals (AF647 dye) and proteins (50 kDa streptavidin) across monolayer choroid plexus cells. The integrity of the tightly bound monolayer was observed by the absence of diffusion or transport of low molecular weight dyes (750 maleimide), 10 kDa dextran polymers, or larger proteins (50 kDa). In the SA conjugate, MTS contains biotin in the core structure. The biotin is then conjugated to a streptavidin protein labeled with a fluorophore. Direct conjugation means that the fluorophore is already covalently linked to MTS on the core (i.e., without biotin / streptavidin linkage).

[0146] The stability of different MTS sequences is important. Figure 22 MTS_CPEC4_V2 is shown to be stable in serum. 80% of the peptides remained intact over 24 hours. Modifications were mapped by MS. MS data indicate that NP-PEGH11 was lost via possible oxidation of methionine or tryptophan.

[0147] The MTS_CPEC4_V2 (SEQ ID NO:1) dimer has been used in in vivo experiments, such as Figure 23 As shown. MTS is distributed throughout the ventricular system, indicating that MTS has crossed from the bloodstream into the CSF. MTS is labeled with dye to visualize its location. The MTS is intravenously injected into the tail vein of the rat. After a specified time, the CSF is separated from the cerebellomedullary cistern. The brain is harvested. After a fixation period, the brain is cut into 1 mm coronal segments (from anterior to posterior). The individual brain slices are imaged using fluorescence to locate areas of peptide accumulation.

[0148] Other lead MTS peptides were examined using transwell transport systems established using rat choroid plexus cells. Figure 24 Eight phage clones were enriched in CSF. The transcytosis of single phage clones on Z310 choroid plexus cells and CSF accumulation in rats were then tested in vitro. Phage clones MTS_Z310-2 (SEQ ID NO:4) and MTS_Z310-5 (SEQ ID NO:7) were subsequently used for further evaluation. Figure 25 The transport of protein cargo across rat choroid plexus epithelial cells by the MTS_Z310-2 and MTS_Z310-5 peptides is shown. 20% of the transport was better than observed using CPEC4. Based on alanine scanning, the underlined portion of MTS_Z310-5 has been identified as the key peptide region responsible for activity.

[0149] The dimer MTS_Z310-5 exists in an amount three times that of MTS_CPEC4_V2. Figure 26 MTS was distributed throughout the ventricular system, indicating that it had crossed from the bloodstream into the CSF. The MTS was labeled with dye to visualize its location. The MTS was intravenously injected into the tail vein of the rat. After a specified time, the CSF was separated from the cerebellomedullary cistern. The brain was harvested. After a fixation period, the brain was cut into 1 mm coronal segments (from anterior to posterior). The individual brain slices were imaged using fluorescence to locate areas of peptide accumulation.

[0150] The dimer MTS_Z310-2 exists in an amount that is 9 times greater than that of MTS_CPEC4_V2. Figure 27MTS was distributed throughout the ventricular system, indicating that it had crossed from the bloodstream into the CSF. The MTS was labeled with dye to visualize its location. The MTS was intravenously injected into the tail vein of the rat. After a specified time, the CSF was separated from the cerebellomedullary cistern. The brain was harvested. After a fixation period, the brain was cut into 1 mm coronal segments (from anterior to posterior). The individual brain slices were imaged using fluorescence to locate areas of peptide accumulation.

[0151] The chemical conjugation of dimers and MTS with cargo can be seen in Figure 28 The general structures of MTS constructs, particularly monomeric and dimer constructs, were revealed using different reactive groups or chemical probes on the core.

[0152] Those skilled in the art will recognize or be able to determine numerous equivalents of specific embodiments of the methods and compositions described herein using no more than routine experiments. These equivalents are intended to be covered by the following claims. sequence list <110> SRI International <120> Molecular transport systems to the central nervous system <130> 37794.0096P1 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 1 Asp Ala Tyr Lys Leu Gln Thr Ser Leu Asp Trp Gln Met Trp Asn Pro 1 5 10 15 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 2 Asp Gly Tyr Lys Leu Gln Thr Ser Leu Asp Trp Gln Met Trp Asn Pro 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 3 Asn Gln Glu Tyr Gln His His Lys Ile Lys Val Arg Pro Ser His Gln 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 4 Phe Pro Ser Trp Thr Ser Lys Asn Gln Gln Trp Thr Asn Gln Arg Gln 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 5 Ala His Met Ser Gln Lys Arg Leu Pro His Gln Val His Gln His Gln 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 6 Ala Gly Asn Lys Tyr Glu Tyr Thr Met His Gln Lys His Asn Lys 1 5 10 15 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 7 Ser Lys Glu Thr Tyr Ser Met Asn Ala Gln Arg Gln His Glu Arg Ser 1 5 10 15 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 8 His Arg Tyr Asp Ala Asp Arg His His Ser Phe Thr Pro Gln Tyr His 1 5 10 15 <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 9 Asn Glu Glu Met His Gln Ala Gln Arg His His Val Gln Trp 1 5 10 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 10 Ala Leu Glu Pro Trp Gly Tyr Lys Gln Val Ile Lys Met Ala Pro Asn 1 5 10 15 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 11 Tyr Ser Met Asn Ala Gln Arg Gln His Glu Arg Ser 1 5 10 <210> 12 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs; molecular transport peptides <400> 12 Tyr Ser Met Asn 1

Claims

1. Use of molecular transport system (MTS) peptides in the preparation of agents for targeting the central nervous system, wherein said MTS peptides consist of the following amino acid sequence: DAYKLQTSLDWQMWNP (SEQ ID NO: 1); or DGYKLQTSLDWQMWNP (SEQ ID NO: 2).

2. The use according to claim 1, wherein the MTS peptide has an N-terminal protecting group.

3. The use according to claim 2, wherein the N-terminal protecting group is an acetyl group.

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

5. The use according to claim 4, wherein the connector is a polyethylene glycol (PEG) connector.

6. The use according to claim 5, wherein the PEG connector is PEG11.

7. The use according to claim 4, wherein the linker is located at the C-terminus of the MTS peptide.

8. The use according to claim 5 or 6, wherein the linker is located at the C-terminus of the MTS peptide.

9. Use of peptides in the preparation of agents for targeting the central nervous system (CNS), wherein said peptides comprise a first molecular transport system (MTS) peptide conjugated to a cargo, said first MTS peptide comprising the following amino acid sequence: DAYKLQTSLDWQMWNP (SEQ ID NO: 1); or DGYKLQTSLDWQMWNP (SEQ ID NO: 2).

10. The use according to claim 9, wherein the cargo is a compound.

11. The use according to claim 9, wherein the cargo is a protein or nucleic acid.

12. The use according to claim 9, wherein the cargo is another peptide.

13. The use according to claim 9, wherein the cargo is an antibody.

14. The use according to claim 10, wherein the compound is an imaging agent.

15. The use according to any one of claims 10-13, wherein the goods are therapeutic agents.

16. The use according to any one of claims 9-14, wherein the peptide further comprises a second MTS peptide.

17. The use according to claim 15, wherein the peptide further comprises a second MTS peptide.

18. The use according to claim 16, wherein the second MTS peptide is the same as the first MTS peptide.

19. The use according to claim 17, wherein the second MTS peptide is the same as the first MTS peptide.

20. Use of the composition in the preparation of an agent for transporting cargo to the central nervous system (CNS), said composition comprising a peptide, said peptide comprising a first molecular transport system (MTS) peptide conjugated to the cargo, said first MTS peptide comprising the following amino acid sequence: DAYKLQTSLDWQMWNP (SEQ ID NO: 1); or DGYKLQTSLDWQMWNP (SEQ ID NO: 2).

21. The use according to claim 20, wherein the cargo is a compound.

22. The use according to claim 20, wherein the cargo is a protein or nucleic acid.

23. The use according to claim 20, wherein the cargo is another peptide.

24. The use according to claim 20, wherein the cargo is an antibody.

25. The use according to claim 21, wherein the compound is an imaging agent.

26. The use according to any one of claims 21-24, wherein the goods are therapeutic agents.

27. The use according to any one of claims 20-25, wherein the composition further comprises a pharmaceutically acceptable carrier.

28. The use according to any one of claims 20-25, wherein the first MTS peptide has an N-terminal protecting group.

29. The use according to claim 28, wherein the N-terminal protecting group is an acetyl group.

30. The use according to any one of claims 20-25 and 29, wherein the peptide comprises a connector between the first MTS peptide and the cargo.

31. The use according to claim 30, wherein the connector is a polyethylene glycol (PEG) connector.

32. The use according to any one of claims 20-25, 29 and 31, wherein the peptide further comprises a second MTS peptide.

33. The use according to claim 32, wherein the second MTS peptide is the same as the first MTS peptide.

34. Use according to any one of claims 20-25, 29, 31 and 33, wherein when the agent is used, one or more of the compositions are administered to a subject in need, wherein the peptide conjugated with the cargo enters the CNS.

35. The use according to claim 34, wherein the first MTS peptide conjugated with the cargo enters the choroid plexus.

36. The use according to claim 34, wherein the peptide enters the cerebrospinal fluid (CSF).

37. The use according to claim 36, wherein the CSF transports the cargo throughout the CNS.

38. The use according to claim 34, wherein the cargo retains its functional activity within the CNS.

39. The use according to claim 34, wherein the administration is intravenous.

40. The use according to any one of claims 9-14, 17-25, 29, 31, 33 and 35-39, wherein when the agent is used, one or more of the peptide or the composition are administered to a subject in need, wherein the agent is a treatment agent for CNS symptoms or injuries.

41. The use according to claim 40, wherein the CNS condition or injury is Parkinson's disease, Alzheimer's disease, glioblastoma, amyotrophic lateral sclerosis, multiple sclerosis, or traumatic brain injury.

42. The use according to claim 40, wherein the CNS disease treatment agent is an antibody or gene therapy.

43. Use of peptides in the preparation of kits for imaging the central nervous system (CNS) of a subject, wherein the peptide comprises a first molecular transport system (MTS) peptide conjugated to a cargo, wherein the first MTS peptide comprises the following amino acid sequence: DAYKLQTSLDWQMWNP (SEQ ID NO: 1); or DGYKLQTSLDWQMWNP (SEQ ID NO: 2); The cargo mentioned therein is an imaging agent.

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