Dual targeting for cell-specific delivery to the central nervous system

Abstract

[Solution] Disclosed are combinations including MGS peptides, MTS peptides, linkers, and any combination thereof. Disclosed are MGS peptides comprising any of the amino acid sequences of SEQ ID NOs: 1-8. Disclosed are MTS peptides comprising any of the amino acid sequences of SEQ ID NOs: 9-14. Disclosed are compositions comprising at least one MGS peptide conjugated to at least one MTS peptide, the MGS peptide comprising the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3); or YAWPASGAWT (SEQ ID NO: 4). In some embodiments, the disclosed compositions further comprise one or more linkers.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 292,370, filed December 21, 2021, and U.S. Provisional Patent Application No. 63 / 412,220, filed September 30, 2022, which are incorporated by reference in their entireties herein.

[0002] The Sequence Listing submitted as an ST.26 file entitled "37794_0099P1.xml", created on December 20, 2022, and having a size of 18,251 bytes, is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). [Background technology]

[0003] Access to the central nervous system (CNS) is a bottleneck in the development of neurotherapeutics. This is due to the blood-brain barrier (BBB), a series of specialized and highly selective cellular barriers that protect the central nervous system (CNS). Although necessary under normal physiology, the BBB prevents many chemicals, such as neurotherapeutics, from entering the brain. As a result, less than 5% of small molecule drugs cross the BBB. Furthermore, new biological therapies, such as antibodies and gene therapies, are essentially excluded from the CNS due to the BBB. Despite the discovery of the BBB more than 100 years ago, no general solution has been generated for transport to the CNS. As a result, many CNS diseases have no treatment options.

[0004] Currently, several approaches are being adopted for delivery to the CNS. One approach is the use of BBB-permeable compounds. The problem with using BBB-permeable compounds is that they tend to have poor biodistribution characteristics (as they often tend to be highly lipid soluble) and off-target effects. Also, there are few BBB-permeable compounds, as less than 5% of small molecules penetrate the BBB. The indications for BBB-permeable drugs are also limited.

[0005] Another approach to accessing the CNS is direct injection into the spinal cord or brain, which is invasive and carries the risk of structural damage to surrounding tissues and increases the risk of infection.

[0006] Alternatively, disruption of the BBB can be used, allowing the mass transport of compounds, cells, and pathogens into the CNS, which can lead to neuronal dysfunction and structural damage.

[0007] Nasal administration has also been used. This method is limited to lipophilic small molecule drugs. Nasal administration has been shown to result in poor distribution throughout the CNS. Furthermore, nasal administration results in variable absorption between doses and patients.

[0008] Receptor-mediated delivery (Trojan horse) methods have also been employed. This method lacks versatility as it is often only effective for a single cargo. Receptors are expressed in multiple tissues, leading to poor uptake into the CNS and toxicity. Furthermore, receptor-mediated delivery lacks cell specificity once transported across the BBB, resulting in inconsistent distribution throughout the CNS.

[0009] There are no known choroid plexus transport agents or alternative methods for transporting cargo across the blood-cerebrospinal fluid barrier, providing a unique opportunity for development to affect delivery of drugs and macromolecules to the CNS. Thus, disclosed herein are compositions and methods for targeting the CNS. Summary of the Invention

[0010] Disclosed are MGS peptides having an amino acid sequence of any of SEQ ID NOs: 1 to 8 shown in Table 1.

[0011] Disclosed herein is an MTS peptide having an amino acid sequence of any of SEQ ID NOs: 9 to 14 shown in Table 2.

[0012] In some embodiments, the linker is [Formula 1] JPEG2025501742000002.jpg21144, where X is a peptide and R is a reactive group capable of chemically reacting with a moiety. In some embodiments, X can be an MGS peptide or an MTS peptide. In some embodiments, this structure is referred to as a dimer core because it can link two peptides.

[0013] In some embodiments, the linker is [Formula 2] JPEG2025501742000003.jpg58144 or [Formula 3] JPEG2025501742000004.jpg37144, where X is a peptide and R is a reactive group that can be chemically reacted with a moiety. In some embodiments, X can be an MGS peptide or an MTS peptide. In some embodiments, this structure is called a tetrameric core because it can link four peptides. Another example of a tetrameric core is [Formula 4] JPEG2025501742000005.jpg46144, where X is a peptide and R is a reactive group capable of chemically reacting with a moiety. In some embodiments, X can be an MGS peptide or an MTS peptide. Disclosed is a composition comprising at least one MGS peptide conjugated to at least one MTS peptide, the MGS peptide having the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4).

[0014] Disclosed is a composition comprising two or more MGS peptides conjugated to two or more MTS peptides, wherein the two or more MGS peptides have the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4).

[0015] A composition is disclosed comprising four MGS peptides conjugated to two MTS peptides, the four MGS peptides having the amino acid sequences of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4), and the two MTS peptides having the amino acid sequences of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0016] Disclosed are compositions comprising an MGS peptide having an amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3), a first linker having (i) at least one reactive group capable of being attached to the C-terminus of the peptide and (ii) at least one additional reactive group capable of chemically reacting with a moiety, a second linker, a third linker having (i) at least one reactive group capable of being attached to the C-terminus of the peptide and (ii) at least one additional reactive group capable of chemically reacting with a moiety, and an MTS peptide having an amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0017] Disclosed are methods of transporting a cargo to the CNS of a subject comprising administering to a subject in need thereof one or more of the compositions disclosed herein, wherein the peptide conjugated to the cargo enters the CNS.

[0018] Disclosed is a method of treating a CNS disorder or injury in a subject comprising administering to a subject in need thereof one or more of the compositions disclosed herein, wherein the cargo is a therapeutic agent for the CNS disorder or injury.

[0019] Disclosed are methods of imaging the CNS of a subject comprising administering to a subject in need thereof one or more of the compositions disclosed herein, wherein the cargo is an imaging agent.

[0020] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

[0021] The accompanying drawings, which are incorporated in and constitute a 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. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows an example of the structure of CH3CO-YAAWPASGAWT (SEQ ID NO:8), MGS2_V4.

[0023] [Diagram 2]Figure 2 shows an example of CH3-CO-GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:5), MGS_NOE3_V2.

[0024] [Diagram 3] 3A and 3B show a dimeric core (A) linker and a tetrameric linker (B) where X is any peptide, particularly MGS or MTS, and R is the attachment point for a cargo such as a dye, imaging agent, therapeutic agent, protein, nucleic acid, carbohydrate, other peptides, lipids, or other chemical modifications.

[0025] [Figure 4] 4A to 4C show examples of MTS peptide structures, where FIG. 4A shows an example of MTS peptide Ac-DAYKLQTSLDWQMWNP, also known as MTS1_V2, FIG. 4B shows an example of MTS peptide Ac-FPSWTSKNQQWTNQRQ, also known as MTS2_V1, and FIG. 4C shows an example of MTS peptide Ac-SKETYSMNAQRQHERS, also known as MTS3_V1.

[0026] [Diagram 5] FIG. 5 shows an example of the structure of the dimeric core modified with a fatty acid; the resulting MTS peptide is called MTS3_V2-2; this modification can be performed on MTS1 and MTS2 as well.

[0027] [Figure 6] Figure 6 shows a schematic of the experimental workflow to resolve MTS trafficking to the CNS in vivo.

[0028] [Figure 7]7A and 7B show that lipidation of MTS3 with C16 fatty acids increases transport to CSF ​​(A) and brain (B), where MTS3_V1-2 is a dimeric Ac-SKETYSMNAQRQHERS (SEQ ID NO:14), MTS3_V2-2 is a dimeric C16 fatty acid modified core with Ac-SKETYSMNAQRQHERS (SEQ ID NO:14), and scMTS3_V2-2 is a dimeric C16 fatty acid modified core with a scrambled MTS3 sequence (scMTS3:RKSAYNQHSSQMREET; SEQ ID NO:15).

[0029] [Figure 8] FIG. 8 shows increased brain accumulation of MTS3_V2-2 compared to the parental MTS3_V1-2 as evidenced by brain sectioning and fluorescence imaging, both versions are conjugated to the same near-infrared dye, circulation times are indicated within each panel, and intensity scale bars are shown kept constant in all images for comparison.

[0030] [Figure 9] 9A-9C show that MGS2_V4-2 is specific to microglial cells and promotes high levels of cellular uptake, FIG. 9A shows HMC3 cells (human microglial cell line) incubated with various concentrations of MGS2_V4-2 at 37° C. for 1 hour, with an EC50 (half maximal uptake) of 7.3 nM, and FIG. 9B shows that uptake of MGS2_V4-2 at 1 hour was determined on HMC3 cells exhibiting either a quiescent or activated phenotype. In both cell conditions, an average of ≈60,000 molecules of MGS2_V4-2 were internalized, indicating that MGS2_V4-2 is effective for transport into microglial cells in both phenotypic conditions; Figure 9C shows that in HMC3 cells, uptake was significantly higher than in other cells of the central nervous system, indicating the specificity of MGS2_V4-2 for microglial cells; all cell lines were incubated with 25 nM MGS2_V4-2 for 1 h at 37°C.

[0031] [Figure 10]FIG. 10 shows that the optimized MGS2_V4-2 is stable in human serum.

[0032] [Figure 11] FIG. 11 shows the structure of the MTS3_V1-2-MGS2_V4-2 chimera, where X=MGS2_V4, X′=MTS3_V1, and R=an attachment point for a dye, imaging agent, therapeutic agent, protein, nucleic acid, other peptide, carbohydrate, and / or lipid.

[0033] [Figure 12] FIG. 12 shows an example of dual targeting in which MTS-MGS chimera (MTS3_V1-2-MGS2_V4-2) is internalized into microglia.

[0034] [Figure 13] 13A-13C show an example of dual targeting where MTS-MGS chimeric agent (MTS3_V1-2-MGS2_V4-2) is internalized into microglia as evidenced by confocal microscopy, single Z slices are shown, scale bar=10 μm.

[0035] [Figure 14]14A-14C show dual targeting of MTS3_V1-2-MGS2_V4-2 chimera to microglia in the CNS, MTS3_V1-2-MGS2_V4-2 conjugate was labeled with Alexa Fluor 647 (red) and injected (0.09 nmol / g) into the lateral tail vein of Sprague Dawley rats, after 2 hours of circulation time, animals were euthanized, brains were fixed by transcardial perfusion, excised, cryoprotected, and cryosectioned in 15 μm sections on a Leica CM1950, images were taken on a Zeiss LSM 800, microglia were stained with Iba-1 (grey) and cell nuclei were stained with Hoechst stain. Stained with 33342 (blue), Figures 14A and 14B depict single z-slice images of the MTS3_V1-2-MGS2_V4-2 conjugate internalized into microglia but not into adjacent non-microglial cells as evidenced by perinuclear staining, and Figure 14C is a 3D rendering of the MTS3_V1-2-MGS2_V4-2 conjugate internalized into microglia but not into surrounding non-microglial cells (magnification of all images = 630x).

[0036] [Figure 15] FIG. 15 shows dual targeting of the MTS3_V1-2-MGS2_V4-2 chimera to microglia in the CNS as demonstrated by flow cytometry.

[0037] [Figure 16] FIG. 16 shows the structure of the MTS3_V1-2-MGS2_V4-4 chimera, where X=MGS2_V4, X′=MTS3_V1, and R=an attachment point for a dye, imaging agent, therapeutic agent, protein, nucleic acid, other peptide, carbohydrate, and / or lipid.

[0038] [Figure 17] FIG. 17 shows the structure of the MTS3_V1-2-MGS2_V4-4 chimera using alternative linkages compared to FIG. 16, where X=MGS2_V4, X'=MTS3_V1 or MTS2_V1, and R=attachment point for dyes, imaging agents, therapeutic agents, proteins, nucleic acids, other peptides, carbohydrates, and / or lipids.

[0039] [Figure 18] Figures 18A and 18B provide examples of siRNA delivery by MGS2-MTS3 chimeras in microglia, where Figure 18A shows the chimera MGS2_V4-4 (tetramer)-MTS3_V1-2 conjugated to test siRNA using click chemistry, and Figure 18B shows the uptake of MGS2_V4-4-MTS3_V1-2 and MGS2_V4-4-MTS3_V1-2-siRNA.

[0040] [Figure 19] FIG. 19 shows the structure of the tetrameric MGS_NOE3_V2-4 core, where X=MGS_NOE3_V2 peptide sequence CH3CO-GFHNVYPYTWGGFSDLMADEI (SEQ ID NO:5) and R=attachment points for dyes, imaging agents, therapeutic agents, proteins, nucleic acids, carbohydrates, lipids, and other peptides.

[0041] [Figure 20] FIG. 20 shows the structure of the MGS_NOE3_V2 peptide sequence, the sequence of which is CH3-CO-GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:5).

[0042] [Figure 21] Figures 21A to 21C show that tetramer MGS_NOE3_V2-4 is specific for neuronal cells and promotes high levels of cellular uptake; Figure 21A shows that MGS_NOE3_V2-4 binds to two neuronal cell lines but not to microglial or astrocytic cell lines; Figure 21B shows that uptake of MGS_NOE3_V2-4 is concentration-dependent with high levels of internalization; and Figure 21C shows that uptake of MGS_NOE3_V2-4 continued for at least 24 hours, reaching intracellular concentrations of ≈1 μM.

[0043] [Figure 22]FIG. 22 shows that the cellular receptors for NOE_NOE3_V2-4 are recycled; chloroquine inhibits receptor recycling and cycloheximide inhibits new protein synthesis.

[0044] [Diagram 23] 23A to 23C show that MGS_NOE3_V2-4 is internalized in neuronal cells, and the scale bar represents 10 μm.

[0045] [Figure 24] Figures 24A-24C show that MGS_NOE3-V2-4 associates with cells of the CNS after intrathecal injection: (A) gating strategy for total brain cells versus debris, (B) gating strategy to exclude cell doublets, and Figure 24C shows the fluorescence intensity of MGS_NOE3-V2-4 internalized in brain cells after intrathecal injection compared to native animals.

[0046] [Diagram 25] FIG. 25 shows the structure of the MTS3_V1-2-MGS_NOE3_V2-4 chimera, where X=mgs_noe3_v2, X′=MTS3_V1, and R=attachment points for dyes, imaging agents, therapeutic agents, proteins, nucleic acids, carbohydrates, lipids, and other peptides.

[0047] [Figure 26]Figures 26A-26C show that the MTS3_V1-2-MGS_NOE3_V2-4 chimera is internalized into HT22 neuronal cells. Figure 26A shows that the MTS3_V1-2-MGS_NOE3_V2-4 chimera, MGS_NOE3_V2-4 or MTS3_V1-2 were incubated with HT22 cells at 200 nM for one hour and peptide uptake data was obtained by quantitative flow cytometry assay. Figure 26B shows that the uptake of the MTS3_V1-2-MGS_NOE3_V2-4 chimera into HT22 cells was assessed over time. Cells were incubated at 200 nM and the average uptake per cell was determined. Figure 26C shows that HT22 cells were incubated with Alexa Fluor 647-labeled MTS3_V1-2-MGS_NOE3_V2-4 chimera at 200 nM for one hour and peptide uptake data was obtained by quantitative flow cytometry assay. nM and incubated at 37 °C for the indicated times. Live cell imaging was performed using Alexa Fluor 488-conjugated wheat germ agglutinin to label cell membranes and Hoechst 33342 to stain nuclei, and representative images are shown.

[0048] [Figure 27] FIG. 27 shows that MTS2_V1-2 binds to the transferrin receptor to access the CNS, while MTS3_V1-2 utilizes a novel, as yet unidentified, cellular receptor for transport.

[0049] [Figure 28] Figures 28A-28E show core architectures with PEG linkers, where a) is a monomeric core with a PEG linker, b) is a dimeric core with a PEG linker, c) is a dimeric core with a maleimide, d) is a tetrameric core with a PEG linker, and e) is a tetrameric core with a maleimide, where R = attachment points for dyes, imaging agents, therapeutic agents, proteins, nucleic acids, other peptides, carbohydrates, and / or lipids, and X = any MGS, MTS, core conjugated with MGS or MTS, and / or linkers.

[0050] [Figure 29]FIG. 29 shows examples of conjugations between a maleimide core (dimer (as shown) or tetramer) and a 2.2 eq peptide (any core architecture (dimer shown)), where X = MGS or MTS, and R = attachment points for dyes, imaging agents, therapeutic agents, proteins, nucleic acids, other peptides, carbohydrates, and / or lipids.

[0051] [Diagram 30] FIG. 30 shows a schematic of the addition of 1.2 eq of maleimide-PEG4-DBCO to 1 eq of peptide (any core architecture (dimer shown)), where X=MGS or MTS.

[0052] [Diagram 31] FIG. 31 shows an example of chimeric peptide synthesis between 1 eq of a peptide (any core architecture (dimer shown)) conjugated with DBCO and 1.2 eq of a peptide (any core architecture (dimer shown)) with modified lysines in the core, where X=MGS or MTS and R=attachment points for dyes, imaging agents, therapeutic agents, proteins, nucleic acids, carbohydrates, other peptides, and / or lipids.

[0053] [Diagram 32] FIG. 32 shows an example of conjugation between 1.2 eq of chimeric peptide (consisting of any core architecture (tetramer / dimer chimera shown)) with free DBCO and 1 eq of siRNA with a free azide, where X=MGS or MTS and R=attachment point for dyes, imaging agents, therapeutic agents, proteins, nucleic acids, other peptides, carbohydrates, and / or lipids. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] The disclosed methods and compositions may be understood more readily by reference to the following detailed description of specific embodiments and examples contained therein, as well as the drawings and accompanying description.

[0055] It is understood that the disclosed methods and compositions are not limited to particular synthetic methods, particular analytical techniques, or particular reagents, unless otherwise specified, and as such may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0056] Disclosed are materials, compositions, and components that can be used in, can be used in combination with, can be used to prepare, and are products of the disclosed methods and compositions. When these and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even if specific references to various individual and collective combinations and permutations of these compounds are not expressly disclosed. Thus, when a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and the combination molecule AD is disclosed as an example, they are considered individually and collectively, even if each is not individually described. Thus, in this example, each combination AE, AF, BD, BE, BF, CD, CE, CF is specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the exemplary combination AD. Similarly, any subset or combination of these is specifically contemplated and disclosed. Thus, for example, subgroups AE, BF, and CE are specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the exemplary combination AD. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any particular embodiment or combination of embodiments of the disclosed method, and each such combination should be specifically contemplated and considered disclosed.

[0057] Headings are provided for convenience only and are not to be construed as limiting the invention in any way. An embodiment shown under any heading or in any portion of this disclosure may be combined with an embodiment shown under the same or other headings or in other portions of this disclosure.

[0058] A.Definition It is understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, as these may vary, and 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 is limited only by the appended claims.

[0059] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to "MGS peptide" includes a plurality of such MGS peptides, and a reference to "a linker" is a reference to one or more linkers and equivalents thereof known to those skilled in the art.

[0060] As used herein, "treatment" refers to administering a composition of the present invention to a subject, such as a human or other mammal (e.g., an animal model) having a disease or condition, 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 embodiments, the disease or condition may be a CNS-related disease or condition, or a CNS disorder or injury. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition, and / or to subjects who show only early signs of the disease, disorder, and / or condition, with the aim of reducing the risk of developing a condition associated with the disease, disorder, and / or condition. In some embodiments, treatment includes delivering one or more of the disclosed compositions to a subject.

[0061] As used herein, "prevention" means minimizing the likelihood of a subject who is predisposed to developing a disease, disorder, or condition developing the disease, disorder, or condition.

[0062] As used herein, the term "subject" refers to a subject of administration, such as a human. Thus, the subject of the disclosed method may be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. The term "subject" also includes pet animals (e.g., cats, dogs, etc.), livestock animals (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). 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. Thus, it is intended to include adults, children, adolescents, newborns, and fetuses, regardless of male or female.

[0063] 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 of the disclosed methods, the "patient" has been diagnosed with a need for treatment prior to the administering step. In some embodiments, patient and subject may be used interchangeably.

[0064] As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, symbols, or words that represent amino acid residues. Amino acid abbreviations used herein are the conventional one-letter codes for amino acids, 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.

[0065] As used herein, "polypeptide" refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is composed of a sequence of amino acids. The term "polypeptide" includes naturally occurring or synthetic molecules.

[0066] Furthermore, as used herein, the term "polypeptide" refers to amino acids linked together by peptide bonds or modified peptide bonds, such as peptide isosteres, and may include modified amino acids other than the 20 gene-encoded amino acids. Polypeptides may be modified either by natural processes, such as post-translational processing, or by chemical modification techniques known in the art. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, the amino terminus, or the carboxyl terminus. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may have many types of modifications. Modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphitidylinositol, disulfide bond formation, demethylation, formation of cysteine ​​or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and the addition of amino acids to proteins via transfer RNA, such as arginylation (see, e.g., T.E. Creighton, Proteins-Structure and Molecular Properties (2nd ed.), W.H. Freeman and Company, New York, 1993; and B.C. Johnson, ed., Posttranslational Covalent Modification of Proteins, Academic Press, 1999). Press, New York, 1983, pp. 1-12).

[0067] The phrase "nucleic acid sequence" as used herein refers to a natural or synthetic oligonucleotide or polynucleotide capable of hybridizing to a complementary nucleic acid by Watson-Crick base pairing, whether single-stranded or double-stranded, sense or antisense, DNA or RNA, or DNA-RNA hybrid. The nucleic acid sequences of the present invention may also contain nucleotide analogs (e.g., BrdU), and non-phosphodiester bonds between nucleosides (e.g., peptide nucleic acid (PNA) or thiodiester bonds). In particular, the nucleic acid sequence includes, but is not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0068] As used herein, an "effective amount" of a composition refers to a sufficient amount of the composition to provide a desired effect. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease (or underlying genetic defect) being treated, the specific compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact "effective amount". However, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0069] As used herein, "selectively binds" means that the MGS or MTS recognizes and physically interacts with its target (e.g., a particular cell type) and does not significantly recognize or interact with other targets.

[0070] The term "percent (%) homology" is used interchangeably herein with the term "percent (%) identity" and refers to the level of identity of a nucleic acid or amino acid sequence 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 the same as 80% sequence identity as determined by a defined algorithm, and thus, a homolog of a given sequence has greater than 80% sequence identity across the given sequence. Exemplary levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity to a given sequence, such as any MTS sequence described herein. Examples of computer programs that may be used to determine identity between two sequences include, but are not limited to, BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN, which are publicly available on the Internet. See also Altschul et al., 1990 and Altschul et al., 1997. Typically, sequence searches are performed using the BLASTN program when evaluating a given nucleic acid sequence against nucleic acid sequences in GenBank DNA Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences translated in all reading frames against amino acid sequences in GenBank Protein Sequences and other public databases. BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0, an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix (see, e.g., Altschul, SF, et al. Nucleic Acids Res. 25:3389-3402, 1997). For example, preferred alignment of selected sequences to determine the "% identity" between two or more sequences is performed using the CLUSTAL-W program in Mac Vector version 13.0.7, operated with default parameters including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM30 similarity matrix.

[0071] Substitutions, deletions, insertions, or any combination thereof may be used to arrive at the final derivative, variant, or analog. Generally, these changes are made to a few nucleotides to minimize the alteration of the molecule. However, in some circumstances, larger changes may be tolerated.

[0072] Generally, the nucleotide identity between individual mutant sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a "mutant sequence" has a certain identity to a parent or reference sequence of the present invention (e.g., a wild-type sequence) and shares a biological function, including but 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 "mutant sequence" can be a sequence that contains one, two, three, or four nucleotide base changes compared to a parent or reference sequence of the present invention, and shares or improves the biological function, specificity, and / or activity of the parent sequence. Thus, a "variant sequence" may have a certain identity to a parent sequence of the invention and share a biological function, including but 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. A 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 a reference sequence (e.g., an MTS sequence).

[0073] "Optional" or "optionally" means that the subsequently described event, circumstance, or material does not have to occur or be present, and the description includes instances in which the event, circumstance, or material occurs or is present as well as instances in which it does not occur or is not present.

[0074] Ranges may be expressed herein as from "about" a particular value and / or to "about" another particular value. Also, when such ranges are expressed, the range from one particular value and / or to another particular value is considered specifically contemplated and deemed disclosed, unless the context specifically indicates otherwise. Similarly, when values ​​are expressed as approximations, it is understood that by using the antecedent "about," the particular value forms another embodiment that is specifically considered and should be considered disclosed, unless the context specifically indicates otherwise. Moreover, the endpoints of each range are understood to be significant both in relation to the other endpoint and independently of the other endpoint, unless the context specifically indicates otherwise. Finally, it is understood that all individual values ​​and subranges of values ​​falling within an expressly disclosed range are considered specifically contemplated and deemed disclosed, unless the context specifically indicates otherwise. The above applies regardless of whether any or all of these embodiments are expressly disclosed in a particular instance.

[0075] 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 belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present methods and compositions, particularly useful methods, devices, and materials are as described. Publications cited herein and the materials to which they refer are specifically incorporated herein by reference. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by prior invention. No admission is made that any document is prior art. The description of the references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. Although a number of publications are referenced herein, it is expressly understood that such reference is not an admission that any of these documents form part of the general knowledge in the art.

[0076] Throughout the description and claims, the word "comprising" and variations of words such as "comprising," "comprising," etc., mean "including, but not limited to," and are not intended to exclude, for example, other additives, ingredients, integers, or steps. Specifically, in a method described as comprising one or more steps or operations, each step is specifically intended to comprise what is recited (unless the step includes a limiting term such as "consisting of"), and each step is not intended to exclude, for example, other additives, ingredients, integers, or steps not recited in that step.

[0077] B. Molecular Guidance System (MGS) Peptides Disclosed herein are MGS peptides. In some embodiments, the MGS peptides may selectively bind to specific cell types. For example, disclosed herein are MGS peptides that can selectively bind to cells of the CNS, such as neurons and microglial cells.

[0078] The present disclosure discloses the use of phage display to screen for neuronal targeting molecular guidance system ("MGS") peptides with high affinity and specificity that enable efficient transport of therapeutic molecules to neuronal cells. This technology allows for rapid and efficient screening of peptide sequences that can be used for neuronal targeting. Neuronal targeting MGS peptides show high affinity to neuronal cells and can be internalized into neuronal cells. Furthermore, neuronal targeting MGS peptides have shown selectivity in brain cells (e.g., astrocytes, microglia).

[0079] The neuronal targeting MGS peptides may be conjugated with small molecules, nucleic acids, and antibodies for therapeutic purposes. In addition, the diverse chemical nature of the peptide sequences allows for modifications to enhance the sensitivity and specificity of neuronal targeting, and combinations with other peptides for dual or multi-targeting (e.g., combinations with blood-brain barrier-crossing peptides). These neuronal targeting peptides also have the ability to induce high neuronal targeting sensitivity and specificity. Thus, these neuronal targeting peptides have potential as induction systems for the treatment of neurological diseases such as Alzheimer's disease and Parkinson's disease.

[0080] Disclosed are MGS peptides comprising the amino acid sequence of any of the sequences set forth in Table 1, SEQ ID NOs:1-8. [Table 1]

[0081] Although Table 1 shows only monomeric MGS peptide sequences, MGS peptides may be used as multimers, such as dimers, tetramers, etc. In some embodiments, the peptide name suffixed with "-2" refers to a dimeric MGS peptide. In some embodiments, the peptide name suffixed with "-4" refers to a tetrameric MGS peptide. For example, MGS_NOE3_V1-2 refers to a dimeric version of the GFHNVYPYTWGGFSDLMADEI (SEQ ID NO:1) sequence. In some embodiments, MGS_NOE3_V1-4 refers to a tetrameric version of the GFHNVYPYTWGGFSDLMADEI (SEQ ID NO:1) sequence. In some embodiments, MGS_NOE3_V2-2 refers to a dimeric version of the Ac-GFNVYPYTWGGFSDLMADEI (SEQ ID NO:5) sequence. In some embodiments, MGS_NOE3_V2-4 refers to the tetrameric version of the Ac-GFNVYPYTWGGFSDLMADEI (SEQ ID NO:5) sequence, and similarly for EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2), MGS_Neuron1_V1-2 is a dimer and MGS_Neuron1_V1-4 is a tetramer. Thus, if MGS_Neuron1_V2, MGS_Neuron2_V1, MGS_Neuron2_V2, MGS2_V3, and MGS2_V4 are monomeric forms of the peptides shown in Table 1, then MGS_Neuron1_V2-2, MGS_Neuron2_V1-2.MGS_Neuron1_V2-2, MGS_Neuron2_V2-2, MGS2_V3-2, and MGS2_V4-2 are dimers, and MGS_Neuron1_V2-4, MGS_Neuron2_V1-4, MGS_Neuron2_V2-4, MGS2_V3-4, and MGS2_V4-4 are tetramers.

[0082] In some embodiments, one or more MGS peptides have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to any of the sequences of SEQ ID NOs:1-8. In some embodiments, one or more MGS peptides have 100% identity in an active portion of the peptide, the active portion being the portion that retains the ability to target CNS cells. Thus, in some embodiments, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any of the MGS peptides occurs outside of the active portion.

[0083] In some embodiments, the MGS peptides may be modified. In some embodiments, the modification of the MGS peptides includes optimizing the peptide or stabilizing the peptide. In some embodiments, the MTS peptides may be optimized. The optimized peptides may be obtained by making modifications to the individual parent peptide sequences. These modifications may be used to identify essential amino acids in the parent sequences required for transport from blood to CSF. These modifications may be obtained by a combination of alanine scanning and truncation of the amino- and C-terminal regions of the parent peptides. PEG can protect the C-terminus of the MGS peptides and provide a spacer between the peptide and the cargo molecule attached via the C-terminal cysteine, enhancing the solubility of the MGS peptides. Modification of the amino terminus with d-amino acids such as acetylation (CH3CO-), d(Leu) can protect against degradation by peptidases in the blood. The optimized peptide length applicable to all MGS peptides is not uniform, and all modifications may be tested to confirm the effect on peptide uptake and stability. Thus, in some embodiments, the MGS peptides may have an N-terminal protecting group. In some embodiments, the N-terminal protecting group can be any that prevents proteases from truncating amino acids from the N-terminus. In some embodiments, the MGS peptides disclosed herein can be N-terminally modified by acetylation. In some embodiments, the N-terminal protecting group is an acetyl group (Ac=CH3CO). In some embodiments, the N-terminal protecting group can be, but is not limited to, PEG, formyl, CH3-(CH)n-CO, a fluorophore, a fatty acid, an alkylamine, an aryl group, a carbohydrate, a sulfonamide, or a carbamate.

[0084] In some embodiments, the MGS peptides disclosed herein may be chemically conjugated to another MGS peptide, a cargo, and / or another linker. In some embodiments, the chemical conjugate may be polyethylene glycol (PEG). Thus, in some embodiments, the MGS peptides disclosed herein may be pegylated. In some embodiments, the number of PEG units may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, or more. In some embodiments, the number of PEG units may be long enough to separate one or more MGS peptides from the cargo so as to prevent steric interference between one or more MGS peptides and the cargo. Thus, in some embodiments, the MGS peptides disclosed herein may further comprise a linker. For example, the linker and the chemical conjugate may be used interchangeably. In some embodiments, the linker is on the C-terminus of the MGS peptide. In one embodiment, the MGS peptides disclosed herein comprise one or more of the sequences of SEQ ID NO:1-8, which may be chemically conjugated at the C-terminus to PEG or another linker as above. In some embodiments, a linker may link two or more MGS peptides. In some embodiments, a linker may be conjugated to a cargo. As an example, an MGS peptide with a linker may comprise the structure shown in FIG. 3, where X is an MGS peptide and R is a reactive group that may be conjugated to a cargo or another linker.

[0085] In some embodiments, the linker can be any described herein, for example, the linker can be of any length that allows for conjugation of the MGS peptide to something else and prevents steric hindrance.

[0086] In some embodiments, the linker comprises a PEG linker, an alkyl linker, a maleimide linker, an amide linker, a peptide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof.

[0087] In some embodiments, examples of MGS peptide structures are shown in Figures 1 and 2. Figure 1 shows the structure of the MGS2_V4 peptide sequence: CH3CO-YAAWPASGAWT (SEQ ID NO:8). Figure 2 shows the structure of the MGS_NOE3_V2 peptide sequence: CH3-CO-GFHNVYPYTWGFSDIDLMADEI (SEQ ID NO:5).

[0088] In some embodiments, the disclosed MGS peptides may be dimeric, and two MGS peptides may be conjugated. FIG. 3A shows an example of the structure of a dimeric MGS core. In some embodiments, the two MGS peptides may be the same MGS peptide or two different MGS peptides. In some embodiments, the two MGS peptides are conjugated via a linker.

[0089] In some embodiments, the disclosed MGS peptides may be tetramers, and four MGS peptides may be conjugated. FIG. 3B shows an example of the structure of a tetrameric MGS core. In some embodiments, the four MGS peptides may be the same MGS peptide or a combination of different MGS peptides. In some embodiments, the four MGS peptides are conjugated via a linker.

[0090] In some embodiments, the reactive groups can be, but are not limited to, carboxylic acids, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (without acetylenic hydrogen), amides and imides, amines, phosphines and pyridines, anhydrides, azos, diazos, azides, hydrazines and azido compounds, carbamates, epoxides, esters, sulfates, phosphates, esters, thiophosphates and borate esters, halogenated organic compounds, isocyanates and isothiocyanates, ketones, oximes, sulfides (organic).

[0091] C. Molecular Transport System (MTS) Peptides Transport of cargo across the blood-cerebrospinal fluid barrier provides a unique opportunity to affect transport of cargo (e.g., drugs and macromolecules) to the CNS. Thus, herein are peptides for targeting the CNS, often referred to as molecular transport system (MTS) peptides.

[0092] Disclosed herein are MTS peptides having the amino acid sequence of any of SEQ ID NOs:9-14 shown in Table 2. [Table 2]

[0093] Although Table 2 shows only monomeric MTS peptide sequences, MTS peptides may be used as multimers, such as dimers. In some embodiments, the peptide name has a "-2" at the end to refer to the dimeric MTS peptide. For example, MTS1_V2-2 refers to the dimeric version of the Ac-DAYKLQTSLDWQMWNP (SEQ ID NO:10) sequence. In some embodiments, MTS2_V1-2 and MTS3_V1-2 are dimeric versions of MTS2_V1 and MTS3_V1, respectively. In some embodiments, when further optimizations, such as lipidation, are added, the next version or "V" of the peptide is referred to. For example, MTS3_V2-2 is the lipidated version of MTS3_V1-2.

[0094] In some embodiments, one or more MTS peptides have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to any of the sequences of SEQ ID NOs:9-14. In some embodiments, one or more MTS peptides have 100% identity in the active portion of the peptide, the active portion being the portion that retains the ability to travel from blood to CSF. Thus, in some embodiments, the at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any of the MTS peptides occurs outside of the active portion.

[0095] In some embodiments, the MTS peptides may be modified, hi some embodiments, modifying the MTS peptides includes optimizing the peptide or stabilizing the peptide.

[0096] In some embodiments, the MTS peptides may be stabilized so that they are intact (e.g., not degraded) during synthesis and / or storage. In some embodiments, the MTS peptides are stabilized by changing a glycine to an alanine. In some embodiments, the glycine at position 2 of SEQ ID NO:9 or 10 can be stabilized by changing it to an alanine.

[0097] In some embodiments, the MTS peptides may be optimized. Optimized peptides can be obtained by making modifications to the individual parent peptide sequences. These modifications can be used to identify essential amino acids in the parent sequences required for transport from blood to CSF. These modifications can be obtained by a combination of alanine scanning and truncation of the amino- and C-terminal regions of the parent peptides. PEG12 can protect the C-terminus of the MTS peptide and provide a spacer between the peptide and the cargo molecule attached via the C-terminal cysteine, enhancing the solubility of the MTS peptide. Modification of the N-terminus of the MTS peptide with acetylation (CH3CO-) and / or d-amino acids such as d(Leu) can protect against degradation by peptidases in the blood. The optimized peptide length applicable to all MTS peptides is not uniform, and all changes can be tested to confirm the effect on peptide uptake and stability.

[0098] In some embodiments, the MTS peptides disclosed herein may have an N-terminal protecting group. In some embodiments, the N-terminal protecting group may be any group that prevents proteases from trimming amino acids from the N-terminus. In some embodiments, the MTS peptides disclosed herein may be N-terminally modified by acetylation. In some embodiments, the N-terminal protecting group is an acetyl group. Thus, in some embodiments, the MTS peptides disclosed herein may be acetylated. In some embodiments, the N-terminal protecting group may be, but is not limited to, PEG, formyl, CH3-(CH)n-CO, a fluorophore, a fatty acid, an alkylamine, a sulfonamide, an aryl group, a carbohydrate, a D-amino acid, or a carbamate.

[0099] In some embodiments, the MTS peptides disclosed herein may be chemically conjugated to another MGS peptide, a cargo, and / or another linker. In some embodiments, the chemical conjugate may be polyethylene glycol (PEG). Thus, in some embodiments, the MTS peptides disclosed herein may be pegylated. In some embodiments, the number of PEG units may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, or more. In some embodiments, the number of PEG units may be long enough to separate one or more MGT peptides from the cargo so as to prevent steric interference between one or more MTS peptides and the cargo. Thus, in some embodiments, the MTS peptides disclosed herein may further comprise a linker. For example, the linker and chemical conjugate may be used interchangeably. In some embodiments, the linker is on the C-terminus of the MTS peptide. In one embodiment, the MTS peptides disclosed herein comprise one or more of the sequences of SEQ ID NO:9-14, which may be acetylated at the N-terminus and chemically conjugated at the C-terminus to PEG or another linker as above. In some embodiments, a linker may link two or more MTS peptides. In some embodiments, a linker may be conjugated to a cargo. As an example, an MTS peptide with a linker may comprise the structure shown in FIG. 3, where X is an MTS peptide and R is a reactive group that may be conjugated to a cargo or another linker.

[0100] In some embodiments, the MTS peptides disclosed herein may be truncated. In some embodiments, the MTS peptides disclosed herein are truncated to remove all amino acids except the active portion of the MTS peptide. In some embodiments, the active portion of the MTS peptide may be used in the disclosed compositions and methods. In some embodiments, the active portion can be determined using techniques known in the art, such as alanine scanning or truncation studies. An active portion of an MTS peptide is one that retains the ability to travel from blood to CSF. For example, SKETYSMNAQRQHERS (SEQ ID NO:13) can be truncated at the N-terminus by up to four amino acids. In some embodiments, the amino acid sequence YSMNAQRQHERS (SEQ ID NO:16) is the active portion of SKETYSMNAQRQHERS (SEQ ID NO:13).

[0101] In some aspects, stabilized or optimized variants of the MTS peptides disclosed herein are disclosed.

[0102] Figure 4 shows examples of MTS peptides. Figure 4A shows an example of MTS peptide Ac-DAYKLQTSLDWQMWNP (SEQ ID NO: 10), also called MTS1_V2. Figure 4B shows an example of MTS peptide Ac-FPSWTSKNQQWTNQRQ (SEQ ID NO: 12), also called MTS1_V2. Figure 4C shows an example of MTS peptide Ac-SKETYSMNAQRQHERS (SEQ ID NO: 14), also called MTS3_V1.

[0103] In some embodiments, the disclosed MTS peptides may be dimeric, and two MGS peptides may be conjugated. FIG. 3A shows an example of the structure of a dimeric MTS core. In some embodiments, the two MGS peptides may be the same MTS peptide or two different MTS peptides. In some embodiments, the two MTS peptides are conjugated via a linker.

[0104] In some embodiments, the reactive groups can be, but are not limited to, carboxylic acids, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (without acetylenic hydrogen), amides and imides, amines, phosphines and pyridines, anhydrides, azos, diazos, azides, hydrazines and azido compounds, carbamates, epoxides, esters, sulfates, phosphates, esters, thiophosphates and borate esters, halogenated organic compounds, isocyanates and isothiocyanates, ketones, oximes, sulfides (organic).

[0105] D. Linker Linkers are disclosed. In some embodiments, the linker can couple or bond two or more MGS peptides, two or more MTS peptides, two or more linkers, or a linker or peptide to a cargo.

[0106] In some embodiments, a linker coupling two or more MGS peptides or two or more MTS peptides may be referred to as a dimeric core (when it binds two peptides) or a tetrameric core (when it binds four peptides). FIG. 3 shows examples of both dimeric and tetrameric core structures. In FIG. 3, the linker comprises PEG12, but in some embodiments, any length of PEG may be used. For example, any of PEG1-PEG30 may be used. In some embodiments, a length of PEG from 1 to 5000 may be used. In some embodiments, any linker may be used in place of PEG12 in the dimeric or tetrameric core.

[0107] Linkers are disclosed that comprise at least one reactive group capable of being attached to the C-terminus of a peptide and at least one additional reactive group capable of chemically reacting with a moiety.

[0108] In some embodiments, the linker has a length of up to PEG 5000. For PEG linkers, in some embodiments, the length of the linker may be all single PEG up to 5000 PEG. In some embodiments, the linker between the peptide and the cargo may be longer than the linker between two MGS or MTS peptides. In some embodiments, the linker comprises 2-4 PEG linkers. In some embodiments, a linker with two PEG linkers may be referred to as a dimer core. In some embodiments, a linker with four PEG linkers may be referred to as a tetramer core.

[0109] In some embodiments, the linker comprises at least two PEG linkers and a reactive group between the at least two PEG linkers, hi some embodiments, the reactive group connects the at least two PEG linkers.

[0110] In some embodiments, the linker comprises an amino acid, a peptide, an alkyl group, a maleimide, a thiol, a hydrazone, or an amide. In some embodiments, the amic acid can be a modified amino acid. For example, the modified amino acid can be a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, or a functionalized aspartic acid. In some embodiments, the linker comprises biotin.

[0111] In some embodiments, the linker is [Formula 5] JPEG2025501742000008.jpg23144, where X is a peptide and R is a reactive group capable of chemically reacting with a moiety. In some embodiments, X can be an MGS peptide or an MTS peptide. In some embodiments, this structure can be referred to as a dimeric core because it can link two peptides.

[0112] In some embodiments, the linker may further comprise a lipid moiety. For example, one of the R groups from the dimeric core may react with a lipid moiety. Thus, in some embodiments, the linker may comprise: [Formula 6] JPEG2025501742000009.jpg47144, where X is a peptide and R is a reactive group capable of chemically reacting with a moiety. In some embodiments, X can be an MGS peptide or an MTS peptide. In some embodiments, X can be an MGS peptide or an MTS peptide.

[0113] In some embodiments, the linker is [Formula 7] JPEG2025501742000010.jpg60144, where X is a peptide and R is a reactive group that can be chemically reacted with a moiety. In some embodiments, X can be an MGS peptide or an MTS peptide. In some embodiments, this structure can be referred to as a tetrameric core because four peptides can be attached. Another example of a tetrameric core is [Formula 8] JPEG2025501742000011.jpg45144, wherein X is a peptide and R is a reactive group capable of chemically reacting with a moiety. In some embodiments, X can be an MGS peptide or an MTS peptide. In some embodiments, the reactive group can be, but is not limited to, carboxylic acids, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (without acetylenic hydrogen), amides and imides, amines, phosphines and pyridines, anhydrides, azos, diazos, azides, hydrazines and azido compounds, carbamates, epoxides, esters, sulfates, phosphates, thiophosphates and borates, halogenated organic compounds, isocyanates and isothiocyanates, ketones, oximes, sulfides (organic).

[0114] In some embodiments, the moiety may be a cargo. In some embodiments, the cargo may be, but is not limited to, a dye, an imaging agent, a therapeutic agent, a protein, a nucleic acid, an amino acid, a peptide, a lipid, an antibody, a radionuclide, a carbohydrate, or a nanoparticle. In some embodiments, the moiety may be a linker. Thus, in some embodiments, a linker comprising a linker is disclosed. For example, when the linker is a tetramer core (i.e., a first linker), the tetramer core may comprise a moiety that is a second linker. In some embodiments, the second linker may comprise the same or different elements as the tetramer core. In some embodiments, the second linker couples the tetramer core (i.e., a third linker) to the dimer core.

[0115] In some embodiments, any of the first, second, and third linkers may comprise an amino acid, a peptide, an alkyl group, a maleimide, a thiol, a hydrazone, a dibenzocyclooctyne, an azide, or an amide.

[0116] E. Composition Disclosed are compositions comprising one or more of the disclosed MGS peptides, MTS peptides, linkers, and / or combinations thereof.

[0117] Disclosed is a composition comprising at least one MGS peptide conjugated to at least one or more MTS peptides, the MGS peptide comprising the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4).

[0118] A composition is disclosed comprising two or more MGS peptides conjugated to two or more MTS peptides, the MGS peptides comprising the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4).

[0119] In some embodiments, the two or more MGS peptides are four MGS peptides. Thus, for example, the four MGS peptides conjugated to at least two MTS peptides include the amino acid sequences of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4).

[0120] A composition is disclosed comprising four MGS peptides conjugated to two MTS peptides, the four MGS peptides comprising the amino acid sequences of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO. 3); or YAWPASGAWT (SEQ ID NO:4); and the two MTS peptides comprising the amino acid sequences of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0121] In some embodiments, two or more MGS peptides are the same MGS peptide. For example, in some embodiments, when there are four MGS peptides in a composition, all four MGS peptides are the same MGS peptide. In some embodiments, at least one MGS peptide is different from the other MGS peptides. In some embodiments, each MGS peptide is different from the others.

[0122] In some embodiments, the two or more MTS peptides are the same MTS peptide. For example, in some embodiments, there are two MTS peptides in the disclosed compositions, and both of the MTS peptides are the same MTS peptide. In some embodiments, there are two MTS peptides in the disclosed compositions, and the two MTS peptides are different from one another.

[0123] In some embodiments, the MGS peptide and / or the MTS peptide has an N-terminal protecting group. In some embodiments, the N-terminal protecting group is an acetyl group.

[0124] In some embodiments, the disclosed compositions may further comprise one or more linkers. In some embodiments, the disclosed compositions may further comprise a first linker. In some embodiments, the first linker is attached to the C-terminus of the MGS peptide. In some embodiments, the first linker may be any linker described herein. In some embodiments, the first linker comprises a PEG linker, an alkyl linker, a maleimide linker, an amide linker, a peptide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the first linker may link the MGS peptide to another MGS peptide, or link the MGS peptide to a cargo, or link the MGS peptide to another linker. In some embodiments, the first linker may comprise one or more of the structures shown in Figure 3. For example, each MGS peptide may be attached to a linker such as PEG (of any length). Each MGS peptide-PEG may then be attached using one or more amino acids, modified amino acids, or linkers capable of creating a dimeric or tetrameric core as described herein.

[0125] In some embodiments, the composition may further comprise a second linker. In some embodiments, the second linker is attached to the first linker. In some embodiments, the second linker may be attached to a third linker. Thus, in some embodiments, the composition further comprises a third linker.

[0126] In some embodiments, the third linker is attached to the second linker. In some embodiments, the third linker is attached to the C-terminus of the MTS peptide. In some embodiments, the third linker can be any linker described herein. In some embodiments, the third linker comprises a PEG linker, an alkyl linker, a maleimide linker, an amide linker, a peptide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the third linker can attach the MTS peptide to another MTS peptide, or attach the MTS peptide to a cargo, or attach the MTS peptide to another linker. In some embodiments, the third linker can comprise one or more of the structures depicted in FIG. 3. For example, each MTS peptide may be attached to a linker such as PEG (of any length). Each MTS peptide-PEG may then be attached using one or more amino acids, modified amino acids, or linkers capable of creating a dimeric or tetrameric core as described herein.

[0127] In some embodiments, the first linker, and / or the second linker, and / or the third linker include a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, a disulfide linker, an amide linker, a peptide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne, 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the first, second, and / or third linker includes a PEG linker. In some embodiments, a PEG linker of any length may be used. For example, any of PEG1 to PEG30 may be used. In some embodiments, a PEG of 1 to 5000 in length may be used.

[0128] In some embodiments, the composition further comprises a cargo. In some embodiments, the cargo may be, but is not limited to, a dye, an imaging agent, a therapeutic agent, a protein, a nucleic acid, an amino acid, a peptide, a lipid, a small molecule, an antibody, a radionuclide, a carbohydrate, or a nanoparticle. For example, in some embodiments, the cargo is an imaging agent, an antibody, and / or an siRNA. In some embodiments, the cargo is attached to a first linker, a second linker, and / or a third linker. Thus, in some embodiments, the disclosed composition may comprise one or more cargoes. In some embodiments, one or more linkers may comprise a cargo.

[0129] In some embodiments, the MTS peptide is any of the MTS peptides described. In some embodiments, the MTS peptide targets the central nervous system. In some embodiments, the MTS peptide comprises the amino acid sequence of any of SEQ ID NOs:9-14. In some embodiments, the MTS peptide comprises the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13). In some embodiments, the MTS peptide may comprise an N-terminal protecting group. In some embodiments, the N-terminal protecting group may be any group that prevents proteases from truncating amino acids from the N-terminus. In some embodiments, the N-terminal protecting group is an acetyl group. In some embodiments, the N-terminal protecting group can be, but is not limited to, PEG, formyl, CH3-(CH)n-CO, an aryl group, a carbohydrate, a D-amino acid, a fluorophore, a fatty acid, an alkylamine, a sulfonamide, or a carbamate.

[0130] In some embodiments, the composition comprises two MTS peptides, hi some embodiments, the two MTS peptides are linked via a dimeric core as described.

[0131] Disclosed are compositions comprising an MGS peptide having an amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); or FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3), a first linker having (i) at least one reactive group capable of being attached to the C-terminus of the peptide and (ii) at least one additional reactive group capable of chemically reacting with a moiety, a second linker, a third linker having (i) at least one reactive group capable of being attached to the C-terminus of the peptide and (ii) at least one additional reactive group capable of chemically reacting with a moiety, and an MTS peptide having an amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0132] In some embodiments, the C-terminus of an MGS peptide comprising the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); or FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3) is attached to a first linker. In some embodiments, the first linker is attached to a second linker. In some embodiments, the second linker is attached to a third linker. In some embodiments, the third linker is attached to the C-terminus of a peptide (MTS) comprising the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0133] Thus, in some embodiments, compositions are disclosed that include an MGS peptide having an amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); or FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3) linked via a reactive group to a first linker, which is linked to a second linker, which is linked to a third linker, which is linked to the C-terminus of a peptide (MTS) having an amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0134] In some embodiments, the composition comprises: [Formula 9] JPEG2025501742000012.jpg98144, where X is an MGS peptide, X' is an MTS peptide, and R is a reactive group capable of chemically reacting with a moiety. In some embodiments, the MGS peptide is any described herein. In some embodiments, the MGS peptide has an amino acid sequence of any of the sequences of SEQ ID NOs: 1-8 shown in Table 1. In some embodiments, the MTS peptide is any described herein. In some embodiments, the MTS peptide has an amino acid sequence of any of the sequences of SEQ ID NOs: 9-14 shown in Table 2.

[0135] In some embodiments, the composition comprises: [Formula 10] JPEG2025501742000013.jpg62144, where X' is an MTS peptide, X is an MGS peptide, and R is a reactive group capable of chemically reacting with a moiety. In some embodiments, the MGS peptide is any described herein. In some embodiments, the MGS peptide has an amino acid sequence of any of the sequences of SEQ ID NOs: 1-8 shown in Table 1. In some embodiments, the MTS peptide is any described herein. In some embodiments, the MTS peptide has an amino acid sequence of any of the sequences of SEQ ID NOs: 9-14 shown in Table 2.

[0136] In some embodiments, the composition comprises: [Formula 11] The peptide has the structure JPEG2025501742000014.jpg41144, where X' is an MTS peptide, X is an MGS peptide, and R is a reactive group capable of chemically reacting with a moiety. In some embodiments, the MGS peptide is any described herein. In some embodiments, the MGS peptide has an amino acid sequence of any of the sequences set forth in SEQ ID NOs: 1-8 shown in Table 1. In some embodiments, the MTS peptide is any described herein. In some embodiments, the MTS peptide has an amino acid sequence of any of the sequences set forth in SEQ ID NOs: 9-14 shown in Table 2.

[0137] In some embodiments, the reactive group may be, but is not limited to, carboxylic acids, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (without acetylenic hydrogen), amides and imides, amines, phosphines and pyridines, anhydrides, azos, diazos, azides, hydrazines and azido compounds, carbamates, epoxides, esters, sulfates, phosphates, esters, thiophosphates and borates, halogenated organic compounds, isocyanates and isothiocyanates, ketones, oximes, sulfides (organic).

[0138] Pharmaceutical compositions are disclosed. Accordingly, the disclosed compositions further comprise a pharma- ceutical acceptable carrier. Disclosed are compositions comprising an MGS peptide having an amino acid sequence of any of the sequences of SEQ ID NOs: 1-8 shown in Table 1, and a pharma- ceutical acceptable carrier. Disclosed are compositions comprising an MTS peptide having an amino acid sequence of any of the sequences of SEQ ID NOs: 9-14 shown in Table 2, and a pharma- ceutical acceptable carrier. Disclosed is a composition comprising four MGS peptides conjugated to two MTS peptides and a pharma- ceutically acceptable carrier, wherein the four MGS peptides have the amino acid sequences of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4), and the two MTS peptides have the amino acid sequences of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0139] 1. Transport of the composition In the methods described herein, the delivery (or administration) of the peptide or composition disclosed herein may be via various mechanisms. As defined above, the present invention may be a composition comprising any one or more peptides described herein, which may be used to create a composition that may also include a carrier, such as a pharma- ceutically acceptable carrier. For example, a pharmaceutical composition is disclosed that comprises a peptide disclosed herein and a pharma- ceutically acceptable carrier.

[0140] For example, the compositions described herein may comprise a pharma- ceutically acceptable carrier. "Pharmaceutically acceptable" means a material or carrier that would be selected to minimize any degradation of the active ingredient and minimize side effects in the subject, as is well known to those skilled in the art. Examples of carriers include dimyristoylphosphatidylcholine (DMPC), phosphate buffered saline, or multivesicular liposomes. For example, PG:PC:cholesterol:peptide or PC:peptide may be used as a carrier in the present invention. Other suitable pharma- ceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.), edited by AR Gennaro, Mack Publishing Company, Easton, 1995. Typically, an appropriate amount of a pharma- ceutical acceptable salt is used in the formulation to make the formulation isotonic. Other examples of pharma- ceutical acceptable carriers are, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution may be about 5 to about 8, or about 7 to about 7.5. Further carriers are sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the composition, the matrices being in the form of, for example, films, stents (implanted in blood vessels during angioplasty), liposomes, or microparticles.It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition to be administered.Most typically, these are standard carriers for administering drugs to humans, including solutions such as sterile water, saline, buffered solutions at physiological pH, and the like.

[0141] Pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives, and the like, so long as the intended activity of the polypeptides, peptides, nucleic acids, and vectors of the present disclosure is not impaired. Pharmaceutical compositions may also include (in addition to the compositions of the present disclosure) one or more active ingredients, such as antibacterial agents, anti-inflammatory agents, anesthetic agents, and the like. Pharmaceutical compositions may be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated.

[0142] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers are water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases.

[0143] Formulations for optical administration may include ointments, lotions, creams, gels, drops, suppositories, powders, liquids, powders, etc. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0144] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders are desirable. Portions of the compositions may be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, and inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl, arylamines, substituted ethanolamines, and the like.

[0145] The disclosed delivery techniques can be used for the disclosed compositions as well as the disclosed nucleic acid sequences and vectors.

[0146] F. Cargo transportation method A method of transporting a cargo to the CNS of a subject comprising administering to a subject in need thereof one or more of the compositions disclosed herein, wherein the peptide bound to the cargo enters the CNS.

[0147] In some embodiments, the cargo may be, but is not limited to, a dye, an imaging agent, a therapeutic agent, a protein, a nucleic acid, an amino acid, a peptide, a lipid, a small molecule, an antibody, a radionuclide, a carbohydrate, or a nanoparticle. For example, in some embodiments, the cargo is an imaging agent, an antibody, and / or a siRNA.

[0148] In some embodiments, the composition, and thus the cargo, enters the choroid plexus. In some embodiments, the composition, and thus the cargo, enters the cerebrospinal fluid (CSF). In some embodiments, the CSF transports the composition, and thus the cargo, throughout the CNS.

[0149] In some embodiments, the MTS peptide enables the composition to cross the blood-brain barrier and enter the CNS. In some embodiments, the MGS peptide targets the cargo to a specific CNS cell. In some embodiments, the CNS cell is a neuron or a microglia.

[0150] In some embodiments, the cargo retains functional activity within the CNS.

[0151] In some embodiments, administration is intravenous or intrathecal. In some embodiments, any delivery method described herein may be used.

[0152] In some embodiments, the composition comprises four MGS peptides conjugated to two MTS peptides, the four MGS peptides having the amino acid sequences of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4), and the two MTS peptides having the amino acid sequences of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0153] The composition may further comprise one or more linkers as described herein. In some embodiments, the disclosed composition may further comprise a first linker. In some embodiments, the first linker is attached to the C-terminus of the MGS peptide. In some embodiments, the first linker may be any linker described herein. In some embodiments, the first linker may include a PEG linker, an alkyl linker, a maleimide linker, an amide linker, a peptide linker, an amide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the first linker may link the MGS peptide to another MGS peptide, or link the MGS peptide to a cargo, or link the MGS peptide to another linker. In some embodiments, the first linker may comprise one or more of the structures shown in Figure 3. For example, each MGS peptide may be attached to a linker such as PEG (of any length). Each MGS peptide-PEG may then be attached using one or more amino acids, modified amino acids, or linkers capable of creating a dimeric or tetrameric core as described herein.

[0154] In some embodiments, the composition may further comprise a second linker. In some embodiments, the second linker is attached to the first linker. In some embodiments, the second linker is further attached to a third linker. Thus, in some embodiments, the composition further comprises a third linker.

[0155] In some embodiments, the third linker is attached to the second linker. In some embodiments, the third linker is attached to the C-terminus of the MTS peptide. In some embodiments, the third linker can be any linker described herein. In some embodiments, the third linker can include a PEG linker, an alkyl linker, a maleimide linker, an amide linker, an aryl linker, a peptide linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the third linker can attach the MTS peptide to another MTS peptide, or attach the MTS peptide to a cargo, or attach the MTS peptide to another linker. In some embodiments, the third linker can comprise one or more of the structures depicted in FIG. 3. For example, each MTS peptide may be attached to a linker such as PEG (of any length). Each MTS peptide-PEG may then be attached using one or more amino acids, modified amino acids, or linkers capable of creating a dimeric or tetrameric core as described herein.

[0156] In some embodiments, the first linker, and / or the second linker, and / or the third linker include a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, a disulfide linker, a peptide linker, an amide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the first, second, and / or third linker includes a PEG linker. In some embodiments, a linker of any length may be used. For example, any of PEG1 to PEG30 may be used. In some embodiments, a PEG of a length of 1 to 5000 may be used.

[0157] In some embodiments, the cargo is attached to the first linker, the second linker, and / or the third linker. Thus, in some embodiments, the disclosed compositions may comprise one or more cargoes. In some embodiments, one or more linkers may comprise a cargo.

[0158] In some embodiments, the MGS peptide, the MTS peptide, or both are N-terminally protected. For example, in some embodiments, the MGS peptide, the MTS peptide, or both are N-terminally acetylated. Thus, in some embodiments, the MGS peptide or the MTS peptide can be any in Table 1 and / or Table 2.

[0159] G. Treatment method Disclosed are methods of treating a CNS disorder or injury in a subject comprising administering to a subject in need thereof one or more of the compositions disclosed herein, wherein the cargo is a therapeutic agent for the CNS disorder or injury.

[0160] In some embodiments, the CNS disorder or injury is Parkinson's disease, Alzheimer's disease, glioblastoma, amyotrophic lateral sclerosis, multiple sclerosis, or traumatic brain injury. Thus, in some embodiments, the CNS disorder therapeutic agent is an antibody (e.g., monoclonal, polyclonal, bispecific), a gene therapy agent, a compound, a nucleic acid sequence, a small molecule, a ribonucleoprotein, or a peptide (or protein). In some embodiments, specific examples of CNS disorder or injury therapeutic agents can be, but are not limited to, N-methyl D-aspartate (NMDA) antagonists, chemotherapeutic agents, glutamate antagonists, or immunomodulatory agents (e.g., immunosuppressants or immunoactivators). In some embodiments, gene therapy allows for the delivery of genetic material encoding therapeutic molecules. In some embodiments, gene therapy involves the administration of a biological drug, including a recombinant nucleic acid, administered to a subject to regulate, repair, replace, add, or delete a genetic sequence for the purpose of treating or curing a disease. In some embodiments, the nucleic acid may be a small RNA, such as, but not limited to, short interfering RNA (siRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA). In some embodiments, the therapeutic agent may be an antibody targeting Aβ peptide / plaque (e.g., Donanemab, Lecanemab), tau tangles, beta secretase, gamma secretase, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE). In some embodiments, nucleic acid therapy may be used to reduce expression of Aβ peptide, tau tangles, beta secretase, gamma secretase, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), CD22.

[0161] In some embodiments, the composition comprises four MGS peptides conjugated to two MTS peptides, the four MGS peptides having the amino acid sequences of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4), and the two MTS peptides having the amino acid sequences of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0162] The composition may further comprise one or more linkers as described herein. In some embodiments, the disclosed composition may further comprise a first linker. In some embodiments, the first linker is attached to the C-terminus of the MGS peptide. In some embodiments, the first linker may be any linker described herein. In some embodiments, the first linker may include a PEG linker, an alkyl linker, a maleimide linker, an amide linker, a peptide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the first linker may link the MGS peptide to another MGS peptide, or link the MGS peptide to a cargo, or link the MGS peptide to another linker. In some embodiments, the first linker may comprise one or more of the structures shown in Figure 3. For example, each MGS peptide may be attached to a linker such as PEG (of any length). Each MGS peptide-PEG may then be attached using one or more amino acids, modified amino acids, or linkers capable of creating a dimeric or tetrameric core as described herein.

[0163] In some embodiments, the composition may further comprise a second linker. In some embodiments, the second linker is attached to the first linker. In some embodiments, the second linker is further attached to a third linker. Thus, in some embodiments, the composition further comprises a third linker.

[0164] In some embodiments, the third linker is attached to the second linker.

[0165] In some embodiments, the third linker is attached to the C-terminus of the MTS peptide. In some embodiments, the third linker may be any linker described herein. In some embodiments, the third linker may include a PEG linker, an alkyl linker, a maleimide linker, an amide linker, a peptide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the third linker may attach the MTS peptide to another MTS peptide, or attach the MTS peptide to a cargo, or attach the MTS peptide to another linker. In some embodiments, the third linker may comprise one or more of the structures depicted in FIG. 3. For example, each MTS peptide may be attached to a linker such as PEG (of any length). Each MTS peptide-PEG may then be attached using one or more amino acids, modified amino acids, or linkers capable of creating a dimeric or tetrameric core as described herein.

[0166] In some embodiments, the first linker, and / or the second linker, and / or the third linker include a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, a disulfide linker, a peptide linker, an amide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the first, second, and / or third linker includes a PEG linker. In some embodiments, a linker of any length may be used. For example, any of PEG1 to PEG30 may be used. In some embodiments, a PEG of a length of 1 to 5000 may be used.

[0167] In some embodiments, the cargo is attached to the first linker, the second linker, and / or the third linker. Thus, in some embodiments, the disclosed compositions may comprise one or more cargoes. In some embodiments, one or more linkers may comprise a cargo.

[0168] In some embodiments, the MGS peptide, the MTS peptide, or both are N-terminally protected. For example, in some embodiments, the MGS peptide, the MTS peptide, or both are N-terminally acetylated. Thus, in some embodiments, the MGS peptide or the MTS peptide can be any in Table 1 and / or Table 2.

[0169] H. Imaging Methods Disclosed is a method of imaging the CNS of a subject, comprising administering one or more of the compositions disclosed herein to a subject in need thereof, wherein the cargo is an imaging agent. In some embodiments, the imaging agent may be, but is not limited to, a dye, a radionuclide, a contrast agent, a fluorescent protein, or a fluorescent molecule. In some embodiments, the imaging agent is bound to a protein, a peptide, or a nucleic acid.

[0170] In some embodiments, the composition comprises four MGS peptides conjugated to two MTS peptides, the four MGS peptides having the amino acid sequences of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3); or YAWPASGAWT (SEQ ID NO:4), and the two MTS peptides having the amino acid sequences of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0171] The composition may further comprise one or more linkers as described herein. In some embodiments, the disclosed composition may further comprise a first linker. In some embodiments, the first linker is attached to the C-terminus of the MGS peptide. In some embodiments, the first linker may be any linker described herein. In some embodiments, the first linker may include a PEG linker, an alkyl linker, a maleimide linker, an amide linker, a peptide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the first linker may link the MGS peptide to another MGS peptide, or link the MGS peptide to a cargo, or link the MGS peptide to another linker. In some embodiments, the first linker may comprise one or more of the structures shown in Figure 3. For example, each MGS peptide may be attached to a linker such as PEG (of any length). Each MGS peptide-PEG may then be attached using one or more amino acids, modified amino acids, or linkers capable of creating a dimeric or tetrameric core as described herein.

[0172] In some embodiments, the composition may further comprise a second linker. In some embodiments, the second linker is attached to the first linker. In some embodiments, the second linker is further attached to a third linker. Thus, in some embodiments, the composition further comprises a third linker.

[0173] In some embodiments, the third linker is attached to the second linker. In some embodiments, the third linker is attached to the C-terminus of the MTS peptide. In some embodiments, the third linker can be any linker described herein. In some embodiments, the third linker can include a PEG linker, an alkyl linker, a maleimide linker, an amide linker, a peptide linker, an amide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the third linker can attach the MTS peptide to another MTS peptide, or attach the MTS peptide to a cargo, or attach the MTS peptide to another linker. In some embodiments, the third linker can comprise one or more of the structures depicted in FIG. 3. For example, each MTS peptide may be coupled to a linker such as PEG (of any length). Each MTS peptide-PEG may then be coupled using one or more amino acids, modified amino acids, or linkers capable of creating a dimeric or tetrameric core as described herein. For example, in some embodiments, PEG may be added to a deprotected lysine (both the side chain ε-amino group and the amino terminus) during Fmoc solid phase peptide synthesis. This provides two free amino groups for PEG attachment, resulting in a dimer. In the case of a tetramer, a fully deprotected lysine may be coupled to another lysine during peptide synthesis. This results in four free amino groups that can be used to attach PEG.

[0174] In some embodiments, the first linker, and / or the second linker, and / or the third linker include a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, a disulfide linker, a peptide linker, an amide linker, an aryl linker, a dibenzooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, an 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the first, second, and / or third linker includes a PEG linker. In some embodiments, a linker of any length may be used. For example, any of PEG1 to PEG30 may be used. In some embodiments, a PEG of a length of 1 to 5000 may be used.

[0175] In some embodiments, the cargo is attached to the first linker, the second linker, and / or the third linker. Thus, in some embodiments, the disclosed compositions may comprise one or more cargoes. In some embodiments, one or more linkers may comprise a cargo.

[0176] In some embodiments, the MGS peptide, the MTS peptide, or both are N-terminally protected. For example, in some embodiments, the MGS peptide, the MTS peptide, or both are N-terminally acetylated. Thus, in some embodiments, the MGS peptide or the MTS peptide can be any in Table 1 and / or Table 2.

[0177] I. Dose Dosing regimens are disclosed that comprise administering to a subject in need thereof one or more of the disclosed compositions or peptides in a single dose, the single dose comprising an amount effective to enter the CNS and target a particular CNS cell type.

[0178] Disclosed is a dosing regimen comprising administering to a subject in need thereof at least two doses of one or more of the disclosed compositions or peptides, each dose being of the same concentration. In some embodiments, each dose after the first dose may be decreased. In some embodiments, each dose after the first dose may be increased.

[0179] In some embodiments, the single dose may be administered consecutively. In some embodiments, the consecutive administration may be over a period of hours, days, weeks, or months. In some embodiments, there may be two or more doses. In some embodiments, the two or more doses may be administered days, weeks, or months apart.

[0180] J.Kit The above materials and other materials can be packaged together in any suitable combination as a kit useful for carrying out or aiding in carrying out the disclosed methods. A given kit is useful if the kit components are designed and adapted for use together in the disclosed methods. For example, disclosed are kits that include one or more of the disclosed MGS peptides, MTS peptides, linkers, or combinations thereof. For example, disclosed are kits that include any of the disclosed compositions.

[0181] A. Example 1: Dual Targeting Figure 5 shows examples of structures of dimeric cores modified with fatty acids. When X is CH3O-SKETYSMNAQRQHERS (SEQ ID NO:14), the structure is called MTS3_V2-2. Similar structures are obtained when X is SEQ ID NO:9 (MTS1), or SEQ ID NO:10 (acetylated MTS1), or SEQ ID NO:11 (MTS2), or SEQ ID NO:12 (acetylated MTS2).

[0182] FIG. 6 shows a schematic of the general experimental flow used to evaluate the transport of the disclosed compositions to the CNS.

[0183] Lipidation of MTS3 with C16 fatty acids increases its transport to the CSF and brain (Figure 7). MTS variants labeled with the near-infrared dye Alexa Fluor 750 were injected into the tail vein of Sprague Dawley rats at 1 μg MTS / kg rat body weight. At the indicated times, CSF was isolated by capillary puncture of the cisterna magna. Figure 7A shows the concentrations of MTS3 variants in isolated CSF after the indicated in vivo circulation times. MTS3_V2, which contains the C16 fatty acid modification, showed 2.2- to 7.6-fold higher CSF concentrations than nonlipidated MTS3_V1-2. To confirm that fatty acids were not promoting transport to the CNS, we lipidated a control peptide, scMTS3_V2-2, which contains the amino acid composition of MTS3 but whose sequence was scrambled. Uptake of MTS3_V2-2 was 9- to 23-fold higher than scMTS3_V2-2, indicating that transport was dependent on the MTS3 sequence and not on the fatty acid modification. Figure 7B shows the concentrations of the same MTS3 variants in brain homogenates after the indicated circulation times. Transport of MTS3_V2-2 into the brain parenchyma was 10-12 times higher than unmodified MTS3_V1-2, which was 17-31 times higher than the scrambled control, scMTS3_V2-2. Concentrations of MTS3_V1-2 were below the detection limit at 24 h. Concentrations in brain tissue were higher than those in CSF, indicating diffusion throughout the brain and slow washout.

[0184] Brain accumulation was enhanced for MTS3_V2-2 compared to parental MTS3_V1-2, as evidenced by brain sectioning and fluorescence imaging (Figure 8). Coronal slices were 1 mm and placed from anterior (top left) to posterior (bottom right). Consistent with the data from brain homogenates, MTS3_V2-2 (C16 fatty acid modified) had increased brain uptake compared to MTS3_V1-2 at all time points. Fluorescent signal was seen throughout the brain, indicating diffusion within the parenchyma.

[0185] Figure 3 shows examples of core structures used in the microglial targeting experiments shown below. Figure 3A shows a dimeric core and Figure 3B shows a tetrameric core. The core structures shown comprised MGS peptides represented as X. The specific MGS peptide used in the experiments described below was MGS2_V4, CH3CO-YAAWPASGAWT. Specifically, the tetrameric core MGS2_V4-4 was used. CH3O-SS

[0186] MGS2_V4-2 was specific to microglial cells and promoted high levels of cellular uptake (Figure 9). Figure 9A shows that MGS2_V4-2 bound with high affinity to and was internalized by microglial cells, and Figure 9B shows that MGS2_V4-2 bound to and was internalized by both resting and activated microglial cells. MGS2_V4-2 was specific to microglial cells compared to neurons, Schwann cells, and astrocytes (Figure 9C).

[0187] Figure 10 shows that the optimized MGS2_V4-2 is stable in human serum. MGS2_V4-2 labeled with Alex Fluor 647 was incubated in human serum at 37°C. At the indicated times, aliquots were removed, serum proteins were precipitated, and the resulting supernatant was analyzed by reverse-phase HPLC monitoring absorbance at 650 nm. Chromatograms at each time point are shown. The retention time of the inactive peptide was 11.864. A new species with a retention time of 10.005 min was observed at 1 h and continued to increase over time. The peaks were integrated to determine the percentage of inactive peptides and the amount of degradation products observed. After 24 h, more than 75% of MGS2_V4-2 remained inactive. Electrospray mass spectrometry was performed on the resulting peptide product with a retention time of 10.005 min. The mass of this new species corresponded to the loss of three amino acids on both branches of MGS2_V4-2.

[0188] Figure 12 shows the general concept of dual targeting to specific cell types of the CNS. The graph in Figure 12 shows an example of the uptake of MGS2_V4-2, MGS3_V1-2-MGS2_V4-2 chimera, and MTS3_V1-2 determined on HMC3 cells. Cells were incubated with the indicated concentrations of each peptide construct labeled with Alexa Fluor 647. After 1 hour of incubation at 37°C, uptake was measured by quantitative flow cytometry. MGS2_V4-2 and MTS3_V1-2-MGS2_V4-2 chimera showed the same EC50 and intracellular uptake, indicating that binding of MTS3_V1-2 does not affect the cellular efficacy of MGS2_V4-2. As expected, MTS3_V1-2 did not show significant intracellular uptake at any of the concentrations tested. Thus, uptake occurred via MGS, but not MTS.

[0189] Figure 13 shows dual targeting of MTS-MGS chimeric agents (MTS3_V1-2-MGS2_V4-2) internalized in microglia as evidenced by confocal microscopy. HMC3 cells were incubated with 25 nM MTS3_V1-2-MGS2_V4-2 conjugates labeled with Alexa Fluor 647. After 1 h, the peptide solution was removed. Alexa Fluor 488-conjugated wheat germ agglutinin was used to label the cell membrane and Hoechst 33342 was used to stain the nuclei. Live cell imaging was performed on a Zeiss LSM 700. Representative single Z-slice images are shown. Cells treated with Alexa Fluor 647 dye alone (FIG. 13A) or a chimeric construct made with a scrambled sequence version of the MGS2 peptide, SAWAGAYPWAT (SEQ ID NO:17) (FIG. 13B), did not show Alexa Fluor 647 staining, indicating cellular uptake. In contrast, MTS3_V1-2-MGS2_V4-2 was internalized into HMC3 cells, as evidenced by pericellular punctate staining (FIG. 13C). This indicated that MGS2_V4-2 mediated cellular internalization into HMC3 cells, consistent with the flow cytometry assay that quantified uptake.

[0190] Figure 14 shows dual targeting of the MTS3_V1-2-MGS2_V4-2 chimera to microglia in the CNS. Staining was observed only in microglia. No staining of microglia was observed with MTS alone, MGS alone, or a scrambled control version of the conjugate.

[0191] Figure 15 shows dual targeting of MTS3_V1-2-MGS2_V4-2 chimeras to microglia in the CNS, as evidenced by flow cytometry. MTS3_V1-2-MGS2_V4-2 conjugates were labeled with Alexa Fluor 647 (red) and injected into the lateral tail vein of Sprague Dawley rats (0.09 nmol / g). After 2 h of circulation time, animals were euthanized and brain tissue was harvested. Brains were dissociated using a Miltenyi Biotec gentleMACS Octo Dissociator. Whole brain cells were incubated with CD11b / c (microglia) Magnetic MicroBeads (Miltenyi Biotec) and separated from the rest of the brain using a Miltenyi QuadroMACS Separator. Microglia were then stained with Iba1 microglia marker. Flow cytometry data were collected on a BD FACSCelesta. Animals treated with the MTS3_V1-2-MGS2_V4-2 conjugate showed a positive shift in microglial cells (IBA1 positive) but not in other CNS cells (IBA1 negative; FIG. 15).

[0192] Figure 16 shows the structure of the MTS3_V1-2-MGS2_V4-4 chimera used in the siRNA delivery experiments shown in Figure 18. Figure 17 shows a similar structure of the MTS3_V1-2-MGS2_V4-4 chimera using alternative linkages.

[0193] An example of siRNA delivery using MGS2-MTS3 chimera in microglia is shown in Figure 18. Figure 18A shows the structure of chimera MGS2_V4-4(tetramer)-MTS3_V1-2 conjugated to test siRNA using click chemistry. Surprisingly, binding to HMC3 cells was lost when siRNA was conjugated to MGS2_V4-2(dimer)-MTS3_V1-2. However, binding was restored when tetramer MGS2_V4-4 was used as the targeting peptide of this multifunctional chimera (shown in panel A). The uptake of MGS2_V4-4-MTS3_V1-2 and MGS2_V4-4-MTS3_V1-2-siRNA is shown in panel B.

[0194] FIG. 19 shows the structure of the tetramer MGS_NOE3_V2-4 used in FIGS. 20-24 demonstrating neuronal cell targeting.

[0195] Figure 20 shows the structure of the MGS_NOE3_V2 peptide sequence. The sequence was CH3-CO-GFHNVYPYTWGGFSDIDLMADEI. MGS_NOE3_V1 had the same sequence but did not contain the N-terminal protecting group. Thus, the structure in Figure 20 was substituted for X in Figure 19.

[0196] Figure 21 shows that tetrameric MGS_NOE3_V2-4 is specific for neuronal cells and promotes high levels of cellular uptake. Monomeric and dimeric MGS_NOE3_V2-4 do not bind. To work, it must be the tetrameric core. Figure 21A shows the uptake of MGS_NOE3_V1-4, which was incubated with the indicated cell lines at 20 nM for 1 hour. The number of molecules of peptide internalized per cell was determined by a quantitative flow cytometry assay. Uptake was significantly higher in two neuronal cell lines, HT22 (mouse hippocampal neurons) and GT1 / 7 (mouse hypothalamic neurons), than in CTX TNA2 (astrocytes from rat frontal cortex), HMC3 (human microglial cell line), H1299 and H640 (both human non-small cell lung carcinoma cells). MGS_NOE3_V1-4 and MGS_NOE3_V2-4 showed 5-6-fold specificity for neurons over astrocytes and 3-fold specificity compared to microglia. Figure 21B shows that uptake of MGS_NOE3_V2-4 into HT22 increased with increasing concentration. Uptake was also assessed for monomeric (MGS_NOE3_V2-1) and dimeric (MGS_NOE3_V2-2) versions, and no uptake was observed in HT22 cells, indicating that it must be the tetrameric core to induce neuronal binding and uptake. Figure 21C shows that uptake of MGS_NOE3_V2-4 increased over time. HT22 cells were incubated with 200 nM MGS_NOE3_V2-4 at 37 °C for the indicated times. Uptake was measured by flow cytometry assay.

[0197] Cellular receptors for MGS_NOE3_V2-4 were recycled (Figure 22). HT-22 cells were incubated with culture medium containing 100 nM MGS_NOE3_V2-4 in the presence of 100 μM chloroquine or 50 μM cycloheximide for the indicated times. Cellular uptake was analyzed by flow cytometry. Cycloheximide, which inhibits new protein synthesis, did not affect the uptake of MGS_NOE3_V2-4, indicating that new protein synthesis is not required for continued uptake. However, chloroquine, which inhibits endosomal acidification and trafficking, significantly reduced uptake from 1 hour of incubation and continued to suppress MGS internalization for up to 24 hours. Endosomal acidification was required for continued uptake of MGS_NOE3_V2-4. Uptake of MGS_NOE3_V2-4 continues over time. Taken together, these data suggest that the intracellular receptors for MGS_NOE3_V2-4 are internalized and then returned to the cell surface where they repeatedly internalize more MGS_NOE3_V2-4.

[0198] MGS_NOE3_V2-4 was internalized in neuronal cells (Figure 23). HT22 cells were incubated with 200 nM MGS_NOE3_V2-4-streptavidin-AlexaFluor647 conjugate at 37°C for the indicated times. AlexaFluor488-conjugated wheat germ agglutinin was used to label the cell membrane and Hoechst 33342 was used to stain the nuclei. Live cell imaging was performed and representative images are shown. Red punctate staining within the cells was observed at 4 hours (Figure 23B) and continued to increase with time (Figure 23C). A control sample containing streptavidin647 but no MGS_NOE3_V2-4 is shown in panel A. The lack of red staining indicates that MGS_NOE3_V2-4 mediated uptake into the cells. The red staining (green staining) located around the cell periphery indicates internalization of MGS_NOE3_V2-4 into the cells.

[0199] Figures 24A-24C show that MGS_NOE3-V2-4 associated with cells of the CNS after intrathecal injection. MGS_NOE3-V2-4 was labeled with Alexa Fluor 647 and injected intrathecally into C57BL / 6 mice. After 1 h of circulating peptide, animals were euthanized and brain tissue was harvested. Whole brains were then dissociated using a Miltenyi Biotec gentleMACS Octo Dissociator. The number of MGS_NOE3_V2-4 molecules internalized into brain cells was analyzed by flow cytometry (BD FACSCelesta).

[0200] FIG. 35 shows the MTS3_V1-2-MGS_NOE3_V2-4 chimera used in the experiments shown in FIGS.

[0201] Figures 26A-26C show that the MTS3_V1-2-MGS_NOE3_V2-4 chimera was internalized into HT22 neuronal cells. Intracellular perforation staining was observed at 1 hour and continued to increase with time. Figure 26A shows a control sample containing Alexa Fluor 647 but no MGS_NOE3_V2-4 and HT22 cells alone. The dual targeting chimera MTS3_V1-2-MGS_NOE3_V2-4 was internalized into HT22 cells as evidenced by staining within the borders of the cell membrane (Figure 26C, bottom right). The uptake of the dual targeting chimera MTS3_V1-2-MGS_NOE3_V2-4 was reduced 3-fold compared to MGS_NOE3_V2-4 alone, but was still significant. Uptake of the dual targeting chimera MTS3_V1-2-MGS_NOE3_V2-4 continued over time, reaching nearly 250,000 molecules / cell (≈250 nM) at 24 h. Taken together, these data indicate that the MTS3_V1-2-MGS_NOE3_V2-4 chimera retains the ability to be internalized into neurons, reaching intracellular levels of ≈250 nM after 24 h, and that this uptake is driven by the MGS_NOE3_V2-4 component of the chimera.

[0202] FIG. 27 shows that MTS2_V1-2 binds to the transferrin receptor to access the CNS, while MTS3_V1-2 utilizes a novel, yet unidentified, cellular receptor for transport. Recombinant human transferrin receptor was adsorbed to a high-binding ELISA plate at 1 μg / mL. After incubation with biotinylated MTS2_V1-2 or MTS3_V1-2 at various concentrations for 1 hour, samples were removed and plates were washed to remove excess peptide. Retained MTS peptide was detected using streptavidin-HRP and TMB reagents. Absorbance was measured at 450 nm and was proportional to the amount of MTS peptide captured by the transferrin receptor. As shown, MTS2_V1-2 bound to human transferrin in a concentration-dependent manner, saturating at higher concentrations. In comparison, MTS3_V1-2 and the scrambled control peptide of MTS2_V1-2 showed no binding. The binding affinity of MTS2_V1-2 for the transferrin receptor was measured by biolayer interferometry using the OctetRED 96 system. Biotinylated MTS2_V1-2 peptide was captured on a streptavidin probe. The k and k rates were measured at various concentrations of human or mouse transferrin receptor to determine the dissociation constant. The Kd of MTS2_V1-2 binding to the transferrin receptor was 210 nM for the human protein and 12 nM for the mouse receptor. Taken together, these data support that the transferrin receptor was the target of MTS2_V1-2. Although the receptor for MTS3_V1-2 remains unknown, the data indicate that it is not the transferrin receptor, and this peptide likely accesses the CNS by a different mechanism than MTS2_V1-2.

[0203] 1. Materials and Methods i. General method for quantifying peptide internalization in cells Cells were incubated with the desired peptide (MGS, MTS, or MGS-MTS conjugate) labeled with 3Alexa Fluor 647 at 37°C in complete medium. Peptide concentrations, incubation times, and cell lines used are indicated in each figure. Peptides were removed and cells were washed three times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4), twice with 0.1 M HCl-glycine pH 2.2 in 0.9% NaCl, and once with a PBS rinse. Cells were removed by trypsinization. Flow cytometry was performed on a BD FACSCelesta and data were analyzed with FlowJo_v10.8. Cells were gated based on forward and side scatter to include only viable cells, and a minimum of 10,000 events were counted. Absolute peptide uptake per cell was determined using Quantum TM A standard curve was generated using Alexa Fluor 647 microspheres. MFI was determined at 50% of the peak height. Internalized molecules per cell were determined by a standard curve relating MESF to MFI and divided by the number of dye molecules / MGS conjugate. GraphPad Prism® was used for nonlinear regression curve fitting to calculate EC50.

[0204] For experiments to determine whether cellular receptors were recycled, cells were treated with peptides in the presence of 100 μM chloroquine or 250 μM cycloheximide. At the indicated time points, cells were washed and analyzed as above.

[0205] ii. CNS cell lines used in uptake studies All cell lines were maintained according to the manufacturer's protocols and media supplies. [Table 3] HMC3 cells were activated with interferon-γ (IFN-γ) (10 ng / ml, 24 h). The resting phenotype was characterized by expression of IBA1 and the endotoxin receptor CD14, but was negative for the astrocyte marker GFAP. Markers of activated microglia, namely MHCII, CD68, and CD11b, were negative in resting HMC3 cells, but their expression was increased after activation.

[0206] iii. In vivo MTS and MTS-MGS targeting experiments MTS peptide (1 μg / g body weight) directly conjugated to Alexa Fluor 750 dye was injected into Sprague-Dawley rats via the lateral tail vein and allowed to circulate for the indicated times (20 min, 1 h, 6 h, 24 h). Ten minutes before the end of each time period, animals were anesthetized and 2 mL of 200x heparinized saline was injected intraperitoneally. To collect CSF, the dorsal neck was bluntly dissected to expose the transparent dura covering the cisterns. A glass micropipette was used to aspirate CSF from the cerebral vesicles at the indicated times. After CSF collection, transcardial perfusion was performed by flushing with 500 mL of ice-cold 1xPBS containing heparin to remove internal blood. Brains were separated from other tissues. Brains were homogenized in lysis buffer (30 mM Tris-HCl, pH 8.0, 0.05% Triton X-100) using a Dounce homogenizer, and the soluble fraction was collected. A standard curve was constructed using MTS peptide conjugated to Alexa Fluor 750, and the amount of dye in the cerebrospinal fluid and brain was measured.

[0207] If the brain was used for tissue sectioning, the initial perfusion was followed by perfusion with 500 mL of ice-cold 5% paraformaldehyde. 1 mm thick coronal sections were made using brain matrix. The slices were imaged with an Odyssey Imager and the fluorescent signal was collected at 800 nm.

[0208] iv. Confocal microscopy: in vitro cell analysis Test cells were seeded in 35 mm confocal microscopy dishes 24 hours prior to treatment. Test peptides labeled with Alexa Fluor 647 were incubated on the cells in culture medium for the indicated times at 37°C. After incubation, cells were washed three times with PBS, twice with 0.1 M HCl-glycine pH 2.2 in 0.9% NaCl, and once with PBS. Alexa Fluor 488-conjugated wheat germ agglutinin was used to label cell membranes and Hoechst 33342 was used to stain nuclei. Microscopy was performed using a Zeiss LSM 700 equipped with a Pln Apo 63x / 1.4 oil DIC III objective. Images were processed using Zen software.

[0209] For peptides that were biotinylated instead of directly labeled with Alexa Fluor 647, peptide-SA-AF647 conjugates were prepared by mixing Alexa Fluor™ 647-conjugated streptavidin and biotinylated peptide at equal molecular ratios in 100 μl of PBS for 30 min at room temperature. After conjugation, 900 μl of culture medium containing biotin (600 nM) was added to the mixture to saturate all binding sites on streptavidin. The cell culture medium was then replaced with the mixture containing peptide. The final peptide-SA-AF647 is shown in each figure. Microscopy was performed as described above.

[0210] v. Confocal Microscopy and Flow Cytometry Analysis for In Vivo Transport Experiments Sprague-Dawley rats were injected with Alexa Fluor 647-labeled MTS, MGS, or MTS-MGS as described above. After 2 h of circulation, animals were euthanized and brains were fixed by transcardial perfusion, harvested, cryoprotected, and cryosectioned at 15 μm sections on a Leica CM1950. Images were taken on a Zeiss LSM 800. Microglia were stained with Iba-1 and cell nuclei were stained with Hoechst 33342.

[0211] For flow cytometry assays, brains were dissociated using a Miltenyi Biotec gentleMACS Octo Dissociator. Total brain cells were incubated with CD11b / c (microglia) Magnetic MicroBeads (Miltenyi Biotec) and separated from the rest of the brain via a Miltenyi QuadroMACS Separator. Microglia were then stained with Iba1 microglia marker. Flow cytometry data were collected by a BD FACSCelesta.

[0212] Alternatively, brains were dissociated using a Miltenyi Biotec gentleMACS Octo Dissociator and all CNS cells were analyzed as a mixture. No enrichment for CNS subpopulations was performed and cells were analyzed directly by flow cytometry. Gating was performed to remove cell debris and cell doublets.

[0213] vi. Serum stability The concentration of a solution of peptide conjugated to a dye (Alexa Fluor 647) was determined by absorbance at 651 nm, and ~14 nmol of peptide was lyophilized. The lyophilized peptide powder was dissolved in 400 μL of human serum (Innovative Research, H6430) to give a 35 μM solution. Samples were incubated at 37 °C and shaken. At each time point (0, 1, 2, 4, 20, 24 h), a 50 μL aliquot was removed from the reaction and 100 μL of absolute ethanol was added to precipitate the serum proteins. Samples were incubated on ice for 15 min and then centrifuged at 12000 rpm for 5 min. Then, 50 μL of the supernatant was removed and diluted with 150 μL of PBS. Finally, 100 μL of each sample was analyzed by analytical RP-HPLC.

[0214] B. Example 2: Reaction Method 28-32 show examples of different reaction methods for making the disclosed peptides and compositions.

[0215] Preparation of resin for dimer core: To synthesize 25 μmol of peptide (MGS2_V2-4), 80 mg of Rink Amide MBHA resin (Gyros Protein Technology, capacity 0.31 mmol / g) was weighed into a 45 mL reaction vessel and transferred to a peptide synthesizer (PurePep Chorus, Gyros Protein Technology) for automated synthesis. The resin was swollen in 6 mL of a 1:1 mixture of N,N-dimethylformamide (DMF, Fisher) / dichloromethane (DCM, Fisher) for 30 min, and then the water was removed.

[0216] Preparation of resin for tetramer core: To synthesize 12.5 μmol of peptide (MGS2_V4-4), 80 mg of Rink Amide MBHA resin (Gyros Protein Technology, capacity 0.31 mmol / g) was weighed into a 45 mL reaction vessel and transferred to a peptide synthesizer (PurePep Chorus, Gyros Protein Technology) for automated synthesis. The resin was swollen in 6 mL of a 1:1 mixture of N,N-dimethylformamide (DMF, Fisher) / dichloromethane (DCM, Fisher) for 30 min, and then the water was removed.

[0217] Manual addition of modified amino acids: The manual coupling cocktail (to resin) typically contained 4.4 eq HCTU (Gyros Protein Technology), 10 eq N-methylmorpholine (NMM, Gyros Protein Technology), 5 eq Oxyma pure (Gyros Protein Technology), and 5 eq modified amino acids including but not limited to Fmoc-S-tert-butylthio-L-cysteine ​​(CHEM-IMPEX INT'L INC), Nα-Fmoc-Nε-azido-L-lysine (CHEM-IMPEX INT'L INC), and Fmoc-Lys(palmitoyl-Glu-OtBu)-OH (BACHEM) dissolved in 1 mL DMF. After 1 h of coupling, the peptide resin was washed twice with 3 mL DMF, twice with 3 mL DCM, and once with 3 mL DMF, mixing for 20 s each.

[0218] Manual addition of linker: contained 16 eq N,N'-diisopropylcarbodiimide (DIC-Gyros Protein Technology), 16 eq N-methylmorpholine (NMM, Gyros Protein Technology), 8 eq Fmoc-amino PEG propionic acid (Polypure), and 8 eq Oxyma pure (Gyros Protein Technology) dissolved in 1 mL DMF. After 2 h of coupling, the peptide resin was washed again (as above).

[0219] Manual addition of maleimidopropionic acid: contained 16 eq N,N'-diisopropylcarbodiimide (DIC-Gyros Protein Technology), 16 eq N-methylmorpholine (NMM, Gyros Protein Technology), 8 eq 3-maleimido-propionic acid (Polypure), and 8 eq Oxyma pure (Gyros Protein Technology) dissolved in 1 mL DMF. After 2 h of coupling, the peptide resin was washed again (as above).

[0220] Automated peptide synthesis: Peptides (MGS2_V2-4 and MGS2_V4-4) were synthesized on a PurePep Chorus using Fmoc solid phase peptide synthesis. Deprotection and coupling steps utilized nitrogen bubbling for mixing. All steps were performed at room temperature.

[0221] Deprotection: Deprotection consisted of two successive treatments (5 min and 10 min) with 2 mL of 20% piperidine (Sigma-Aldrich) in 0.1 M Oxyma pure (Gyros Protein Technology) / DMF solution. Deprotection was followed by washing (as above).

[0222] Coupling: The coupling steps included 1 mL of 0.25 M Fmoc-amino acid (Gyros Protein Technology) in 0.25 M Oxyma pure / DMF solution, 0.5 mL of 0.44 M HCTU (Gyros Protein Technology) L in DMF, and 0.5 mL of 1 M N-methylmorpholine (NMM, Gyros Protein Technology) in DMF. After 30 min and 1 h of successive couplings, the peptide resin was washed again (as above). After all coupling steps were completed, the peptide resin was deprotected and washed in preparation for acetylation of the N-terminal amine.

[0223] Acetylation: Acetylation of the N-terminal amine is The reaction consisted of two 20 min treatments with a 2 mL solution of 0.2 mL acetic anhydride (Fisher), 0.2 mL NMM, and 1.6 mL DMF. Acetylation was followed by washing four times with 3 mL DMF and eight times with 3 mL DCM. The peptide resin was then dried in vacuum on a Prelude for 1 h.

[0224] Cleavage: The peptide resin was transferred to a 5 mL filter syringe and cleaved with 5 mL cleavage cocktail of 92.5% trifluoroacetic acid (TFA, Fisher), 5% triisopropylsilane (Sigma-Aldrich), and 2.5% MQ-water. The cleavage mixture was stirred for 2-3 h, poured into cold diethyl ether (Fisher), and shaken vigorously to precipitate the peptide. The mixture was centrifuged at 2000 rpm for 2 min at room temperature, and the supernatant was decanted. The crude peptide pellet was washed twice with fresh diethyl ether, dried in a fume hood, and dried overnight in a vacuum desiccator.

[0225] Crude peptide preparation and purification by RP-HPLC: Crude peptide was weighed and dissolved in a solution of 20-40% acetonitrile (Fisher), 80-60% MQ-water, and 0.1% TFA. The peptide solution was vortexed, sonicated, and stirred for 1 h. After dissolution, the peptide solution was filtered through a 0.2 mm filter syringe and prepared for RP-HPLC and analysis. Purification of the peptide was performed using a Waters Prep LC 2767 system with 0.1% TFA in MQ-water (buffer A) and 0.1% TFA in acetonitrile (buffer B) as mobile phases. Each injection was performed at room temperature using a Phenomenex Jupiter 5mm C4 300A column (200x212mm) at a flow rate of 10mL / min with a 20-70% Buffer B profile (0-3min 20%, 3-25min 20-70%, 25-30min 90%, 30-35min 20%). Portions were collected, analyzed by analytical RP-HPLC and ESI-MS to assess purity, and lyophilized.

[0226] Conjugation of the maleimide core with peptide (MGS or MTS): The lyophilized maleimide core was weighed and dissolved in 3M GuHCl / PBS. The lyophilized MGS / MTS peptides were then dissolved separately in 3M GuHCl / PBS and the pH of the solutions was determined and adjusted (if necessary) to ∼7. The solutions were combined to give a 1.0:2.2 molar eq. maleimide core to peptide ratio. The reaction was purified by preparative RP-HPLC (Agilent 1260) and then stirred for 3 h. The peptide products were collected, characterized by analytical RP-HPLC and ESI-MS, and lyophilized (Figure 29).

[0227] Addition of DBCO: The lyophilized peptide was dissolved in 3 M guanidinium chloride (GuHCl, Sigma-Aldrich) in PBS. The pH of the solution was determined and adjusted (if necessary) to ~6.8. Then, 1.2 eq. of Maleimide-PEG4-DBCO (Sigma-Aldrich), also dissolved in 3 M GuHCl / PBS, was added to the peptide solution. The reaction was purified by preparative RP-HPLC (Agilent 1260) and then stirred for 30 min. The peptide product was collected, characterized by analytical RP-HPLC and ESI-MS, and lyophilized (Figure 30).

[0228] Chimeric peptide synthesis: The lyophilized peptides with DBCO conjugated (MGS or MTS) and with Nα-Fmoc-Nε-azido-L-lysine incorporated into the core (MGS or MTS) were weighed and dissolved separately in 3 M GuHCl / PBS. The pH of each solution was determined and adjusted (if necessary) to ∼7. The solutions were combined to give a 1.0:2.2 molar eq. ratio of DBCO conjugated peptide to Nα-Fmoc-Nε-azido-L-lysine conjugated peptide. The reactions were stirred overnight and then purified by preparative RP-HPLC (Agilent 1260). The peptide products were collected, characterized by analytical RP-HPLC and ESI-MS, and lyophilized (Figure 31).

[0229] Conjugation of siRNA with chimeric peptide: Chimeric peptides (of any core structure) with free DBCO were dissolved in nuclease-free PBS (NF-PBS) and the pH was determined and adjusted (if necessary) to ~7. siRNAs incorporating azides at either the 5' or 3' end were then dissolved in NF-PBS and concentrations were determined by measuring absorbance at 260 nm. The solutions were combined to give a 1:1.2 molar eq. ratio of siRNA to peptide. Reactions were stirred for 3-7 days and then purified by analytical RP-HPLC (Agilent 1220). Portions were collected, mixed, characterized by analytical RP-HPLC, and lyophilized (Figure 32).

[0230] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein, which equivalents are intended to be encompassed by the following claims.

Claims

1. A peptide having the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); or FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3).

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

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

4. The peptide of claim 1 , further comprising a linker.

5. 5. The peptide of claim 4, wherein the linker comprises a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, a peptide linker, an amide linker, an aryl linker, a dibenzocyclooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof.

6. The peptide of claim 5 , wherein the PEG linker is PEG12 or PEG14.

7. The peptide of claims 4 to 6, wherein the linker is attached to the C-terminus of the peptide.

8. at least one molecular guide sequence (MGS) peptide conjugated to at least one molecular transport sequence (MTS) peptide; The MGS peptide has the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3); or YAAWPASGAWT (SEQ ID NO: 4). composition.

9. two or more molecular targeting sequence (MGS) peptides conjugated to two or more molecular transport sequence (MTS) peptides; The MGS peptide has the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3); or YAAWPASGAWT (SEQ ID NO: 4). composition.

10. The composition of claim 9 , wherein the two or more MGS peptides are four MGS peptides.

11. The composition of claim 10 , wherein the two or more MGS peptides are the same MGS peptide.

12. The composition of claims 9-11, wherein the two or more MTS peptides are the same MTS peptide.

13. The composition of claims 8-12, wherein the MGS peptide has an N-terminal blocking group.

14. The composition of claim 13 , wherein the N-terminal protecting group is an acetyl group.

15. The composition of claims 8-14, further comprising a first linker.

16. The composition of claim 15 , wherein the first linker is attached to the C-terminus of the MGS peptide.

17. The composition of claims 15 and 16, further comprising a second linker.

18. The composition of claim 17 , wherein the second linker is attached to the first linker.

19. 19. The composition of claim 17 or 18, further comprising a third linker.

20. The composition of claim 19 , wherein the third linker is attached to the second linker.

21. 21. The composition of claims 17-20, wherein the third linker is attached to the C-terminus of the MTS peptide.

22. 22. The composition of claims 15-21, wherein the first linker, and / or the second linker, and / or the third linker comprises a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, a disulfide linker, a peptide linker, an amide linker, an aryl linker, a dibenzocyclooctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof.

23. The composition of claim 22 , wherein the linker comprises a PEG linker.

24. 24. The composition of claims 8-23, further comprising a cargo.

25. 25. The composition of claim 24, wherein the cargo is attached to the first linker, the second linker, and / or the third linker.

26. 26. The composition of claim 24 or 25, wherein the cargo is a nucleic acid sequence, a peptide, a protein, or a small molecule.

27. 27. The composition of claims 24 to 26, wherein the cargo is an imaging agent, an antibody, and / or an siRNA.

28. 28. The composition of claims 8-27, wherein the MTS peptide targets the central nervous system.

29. 29. The composition of claims 8-28, wherein the MTS peptide has the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

30. 30. The composition of claims 8-29, wherein the MTS peptide comprises an N-terminal blocking group.

31. The composition of claim 30 , wherein the N-terminal protecting group is an acetyl group.

32. comprising four molecular guide sequence (MGS) peptides conjugated to two molecular transport sequence (MTS) peptides; The MGS peptide has the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3); or YAAWPASGAWT (SEQ ID NO: 4); The MTS peptide has the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO: 9); FPSWTSKNQQWTNQRQ (SEQ ID NO: 11); or SKETYSMNAQRQHERS (SEQ ID NO: 13). composition.

33. 33. The composition of claim 32, wherein the four MGS peptides are the same or different MGS peptides.

34. The composition of claim 32 , wherein the two MTS peptides are the same or different MTS peptides.

35. 35. The composition of claims 32-34, wherein the MGS peptide has an N-terminal blocking group.

36. The composition of claim 35 , wherein the N-terminal protecting group is an acetyl group.

37. 37. The composition of claims 32-36, further comprising a first linker.

38. The composition of claim 37 , wherein the first linker is attached to the C-terminus of the MGS peptide.

39. 39. The composition of claim 37 or 38, further comprising a second linker.

40. 40. The composition of claim 39, wherein the second linker is attached to the first linker.

41. 41. The composition of claim 39 or 40, further comprising a third linker.

42. 42. The composition of claim 41, wherein the third linker is attached to the second linker.

43. 43. The composition of claim 41 or 42, wherein the third linker is attached to at least one C-terminus of the MTS peptide.

44. 44. The composition of claims 37-43, wherein the first linker and / or the second linker comprises a polyethylene glycol (PEG) linker.

45. The composition of claim 44, wherein the PEG linker is PEGPEG12 or PEG14.

46. 46. ​​The composition of claims 32-45, further comprising a cargo.

47. 47. The composition of claim 46, wherein the cargo is attached to the first linker, the second linker, and / or the third linker.

48. 48. The composition of claim 46 or 47, wherein the cargo is a nucleic acid sequence, a peptide, a protein, or a small molecule.

49. The composition of claim 48, wherein the cargo is an imaging agent, an antibody, and / or an siRNA.

50. 50. The composition of claims 32-49, wherein the MTS peptide targets the central nervous system.

51. 51. The composition of claims 32-50, wherein the MTS peptide comprises an N-terminal blocking group.

52. 52. The composition of claim 51, wherein the N-terminal protecting group is an acetyl group.

53. Administering one or more of the compositions of claims 24-31 or 46-52 to the subject in need thereof; The peptide conjugated to the cargo enters the CNS. A method of delivering said cargo to the CNS of said subject.

54. The method of claim 53, wherein the composition enters the choroid plexus.

55. 55. The method of claim 53 or 54, wherein the composition enters the cerebrospinal fluid (CSF).

56. 56. The method of claim 55, wherein the CSF transports the composition throughout the CNS.

57. 57. The method of claims 53-56, wherein the MGS targets the cargo to specific CNS cells.

58. 58. The method of claim 57, wherein the CNS cell is a neuron or a microglia.

59. 60. The method of claims 53-58, wherein the cargo retains functional activity within the CNS.

60. 60. The method of claims 53-59, wherein administration is intravenous or intrathecal.

61. Administering one or more of the compositions of claims 24-31 or 46-52 to a subject in need thereof, The cargo is a therapeutic agent for a CNS disorder or injury. Methods of Treating the CNS Disorder or Injury in a Subject

62. 62. The method of claim 61, wherein the CNS disorder or injury is Parkinson's disease, Alzheimer's disease, glioblastoma, amyotrophic lateral sclerosis, multiple sclerosis, or traumatic brain injury.

63. 63. The method of claim 61 or 62, wherein the CNS disorder treatment is an antibody or gene therapy.

64. Administering one or more of the compositions of claims 24-31 or 46-52 to a subject in need thereof, The cargo is an imaging agent. A method of imaging the CNS of the subject.

65. 65. The method of claims 61-64, wherein the composition enters the choroid plexus.

66. 66. The method of claims 61-65, wherein the composition enters the cerebrospinal fluid (CSF).

67. 67. The method of claim 66, wherein the CSF transports the composition throughout the CNS.

68. 68. The method of claims 61-67, wherein the MGS targets the cargo to specific CNS cells.

69. 69. The method of claim 68, wherein the CNS cell is a neuron or a microglia.

70. 70. The method of claims 61-69, wherein the cargo retains functional activity within the CNS.

71. 71. The method of claims 61-70, wherein administration is intravenous or intrathecal.

72. (i) at least one reactive group capable of being attached to the C-terminus of a peptide; (ii) at least one additional reactive group capable of chemically reacting with the moiety; and A linker comprising:

73. 73. The linker of claim 72, wherein the linker further comprises a lipid moiety.

74. 74. The linker of claim 72 or 73, wherein the linker comprises two to four PEG linkers and a reactive group between at least two of the PEG linkers.

75. 75. The linker of claims 72-74, wherein the linker comprises an amino acid, a peptide, an alkyl group, a maleimide, a thiol, a hydrazone, and an amide.

76. 76. The linker of claim 75, wherein the amino acid is a modified amino acid.

77. 77. The linker of claim 76, wherein the modified amino acids are functionalized lysine, functionalized cysteine, glutamic acid, and aspartic acid.

78. The linker is [Formula 12] With the structure of X is a peptide; R is a reactive group capable of chemically reacting with a moiety; 78. A linker according to claims 72 to 77.

79. The linker is [Formula 13] With the structure of X is a peptide; R is a reactive group capable of chemically reacting with a moiety; 78. A linker according to claims 72 to 77.

80. The linker is [Formula 14] With the structure of X is a peptide; R is a reactive group capable of chemically reacting with a moiety; 78. A linker according to claims 72 to 77.

81. The linker is [Formula 15] With the structure of X is a peptide; R is a reactive group capable of chemically reacting with a moiety; 78. A linker according to claims 72 to 77.

82. 82. The linker of claims 72-81, wherein the reactive group is carboxylic acid, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (without acetylenic hydrogen), amides and imides, amines, phosphines and pyridines, anhydrides, azos, diazos, azides, hydrazines and azido compounds, carbamates, epoxides, esters, sulfates, phosphates, esters, thiophosphates and borates, halogenated organic compounds, isocyanates and isothiocyanates, ketones, oximes, sulfides (organic).

83. the moiety is a cargo, The cargo is a dye, an imaging agent, a therapeutic agent, a protein, a nucleic acid, a peptide, or a lipid; 83. A linker according to claims 71 to 82.

84. The linker of claims 72 to 83, wherein the moiety is a linker.

85. a. a peptide (MGS) having the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); or FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3); b. a first linker comprising (i) at least one reactive group capable of being attached to the C-terminus of a peptide and (ii) at least one additional reactive group capable of chemically reacting with a moiety; c. a second linker; d. a third linker comprising (i) at least one reactive group capable of being attached to the C-terminus of the peptide and (ii) at least one additional reactive group capable of chemically reacting with a moiety; e. A peptide (MGS) having the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO: 9); FPSWTSKNQQWTNQRQ (SEQ ID NO: 11); or SKETYSMNAQRQHERS (SEQ ID NO: 13); A composition comprising:

86. 86. The composition of claim 85, wherein the C'-terminus of the peptide (MGS) having the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); or FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3) is linked to the first linker.

87. 87. The composition of claim 85 or 86, wherein the first linker is attached to the second linker.

88. 88. The composition of claims 85-87, wherein the second linker is attached to the third linker.

89. 89. The composition of claims 85-88, wherein the third linker is attached to the C-terminus of the peptide (MTS) having the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO: 9); FPSWTSKNQQWTNQRQ (SEQ ID NO: 11); or SKETYSMNAQRQHERS (SEQ ID NO: 13).

90. 90. The composition of claim 85-89, comprising a peptide (MGS) comprising the amino acid sequence of GFHNVYPYTWGGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); or FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3), attached to a first linker via a reactive group.

91. The composition comprises: [Formula 16] With the structure of X' is a peptide having the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO: 9); FPSWTSKNQQWTNQRQ (SEQ ID NO: 11); or SKETYSMNAQRQHERS (SEQ ID NO: 13); X is a peptide having the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3); or YAAWPASGAWT (SEQ ID NO: 4); R is a reactive group capable of chemically reacting with a moiety; 91. The composition of claims 85 to 90.

92. The composition comprises: [Formula 17] With the structure of X' is a peptide having the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO: 9); FPSWTSKNQQWTNQRQ (SEQ ID NO: 11); or SKETYSMNAQRQHERS (SEQ ID NO: 13); X is a peptide having the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3); or YAAWPASGAWT (SEQ ID NO: 4); R is a reactive group capable of chemically reacting with a moiety; 91. The composition of claims 85 to 90.

93. The composition comprises: [Formula 18] With the structure of X' is a peptide having the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO: 9); FPSWTSKNQQWTNQRQ (SEQ ID NO: 11); or SKETYSMNAQRQHERS (SEQ ID NO: 13); X is a peptide having the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO: 2); FQHNPFPYTYSMEDTDVEIK (SEQ ID NO: 3); or YAAWPASGAWT (SEQ ID NO: 4); R is a reactive group capable of chemically reacting with a moiety; 91. The composition of claims 85 to 90.

94. 94. The composition of claims 85-93, wherein the MTS peptide has an N-terminal blocking group.

95. 95. The composition of claim 94, wherein the N-terminal protecting group is an acetyl group.

96. 94. The composition of claims 85-93, wherein the MTS peptide has an N-terminal blocking group.

97. 97. The composition of claim 96, wherein the N-terminal protecting group is an acetyl group.

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