Dual targeting for cell-specific transport 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 hereby incorporated by reference in their entirety.

[0002] Submitted as an ST.26 file named "37794_0099P1.xml" created on December 20, 2022, the array list created on December 20, 2022 and having a size of 18,251 bytes is hereby incorporated by reference in accordance with 37 C.F.R. § 1.52(e)(5).

Background Art

[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 CNS. Although necessary under normal physiology, the BBB prevents the entry of many chemicals, such as neurotherapeutics, into the brain. As a result, less than 5% of small molecule drugs cross the BBB. Furthermore, new biological therapies such as antibodies and gene therapy are essentially excluded from the CNS due to the BBB. Despite being discovered over 100 years ago, no general solution for transport to the CNS has been produced. Therefore, many CNS diseases have no treatment options.

[0004] Currently, several approaches are being adopted for transport to the CNS. One approach is the use of BBB-permeable compounds. The problems with using BBB-permeable compounds are that they tend to have poor in vivo distribution characteristics (as they tend to be highly lipophilic in many cases) and tend to have off-target effects. Also, since less than 5% of small molecules cross the BBB, there are few BBB-permeable compounds. 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. This method is invasive, has a risk of causing structural damage to surrounding tissues, and also increases the risk of infection.

[0006] Also, disruption of the BBB is used as another method. This method enables the mass transport of compounds, cells, and pathogens into the CNS. This method may cause dysfunction and structural damage to nerve cells.

[0007] Furthermore, nasal administration is also used. This method is limited to lipophilic low-molecular drugs. Nasal administration has been shown to have poor distribution across the CNS. Additionally, nasal administration shows variable absorption between doses and patients.

[0008] Also, the receptor-mediated transport (Trojan horse) method is employed. This method often lacks versatility as it is only effective for a single cargo. Since receptors are expressed in multiple tissues, uptake into the CNS is poor and it causes toxicity. Furthermore, once transported across the BBB, receptor-mediated transport lacks cell specificity and the distribution across the CNS is not uniform.

[0009] There is no known choroid plexus transporter or alternative method for transporting cargo across the blood-brain-cerebrospinal fluid barrier, providing an unprecedented development opportunity to affect the transport of drugs and macromolecules into the CNS. Accordingly, the present specification discloses compositions and methods targeting the CNS. SUMMARY OF THE INVENTION

[0010] Disclosed is an MGS peptide comprising an amino acid sequence of any one of the sequences of SEQ ID NOs: 1-8 shown in Table 1.

[0011] Disclosed herein is an MTS peptide. Disclosed is an MTS peptide comprising an amino acid sequence of any one of the sequences of SEQ ID NOs: 9-14 shown in Table 2.

[0012] In some embodiments, the linker is [Formula 1] JPEG0007684523000001.jpg21144 having the structure, where X is a peptide and R is a reactive group that can chemically react with a moiety. In some embodiments, X may be an MGS peptide or an MTS peptide. In some embodiments, this structure can link two peptides and is thus referred to as a dimer core.

[0013] In some embodiments, the linker is [Formula 2] JPEG0007684523000002.jpg58144 or [Formula 3] JPEG0007684523000003.jpg37144 having the structure, where X is a peptide and R is a reactive group that can chemically react with a moiety. In some embodiments, X may be an MGS peptide or an MTS peptide. In some embodiments, this structure can link four peptides and is thus referred to as a tetramer core. Another example of a tetramer core is [Formula 4] JPEG0007684523000004.jpg46144 having the structure, where X is a peptide and R is a reactive group that can chemically react with a moiety. In some embodiments, X may 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, where 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).

[0014] A composition is disclosed that comprises two or more MGS peptides conjugated to two or more MTS peptides, wherein the two or more MGS peptides comprise 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).

[0015] A composition is disclosed that comprises four MGS peptides conjugated to two MTS peptides, wherein the four MGS peptides comprise 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), and the two MTS peptides comprise the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0016] A composition is disclosed that comprises 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), a first linker comprising (i) at least one reactive group capable of binding to the C-terminus of the peptide and (ii) at least one additional reactive group capable of reacting chemically with a moiety, a second linker, a third linker comprising (i) at least one reactive group capable of binding to the C-terminus of the peptide and (ii) at least one additional reactive group capable of reacting chemically with a moiety, and an MTS peptide comprising the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0017] Disclosed is a method of transporting cargo to the CNS of a subject, comprising administering to the 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 the 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 is a method of imaging the CNS of a subject, comprising administering to the 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 methods and compositions are described in part in the following description, understood in part from the description, or can be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions are realized and achieved by the elements and combinations particularly pointed out in the appended claims. It is understood that the above general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claimed invention.

[0021] The accompanying drawings, which are incorporated herein and constitute a part hereof, illustrate several embodiments of the disclosed methods and compositions and, together with the specification, serve to explain the principles of the disclosed methods and compositions.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

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

[0055] The disclosed methods and compositions are not limited to specific synthesis methods, specific analytical techniques, or specific reagents, unless otherwise specified, and thus it is understood that they can vary. It is also understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0056] Materials, compositions, and components that are usable in, combinable with, preparable by, and are products of the disclosed methods and compositions are disclosed. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, each individual and collective combination and permutation of these compounds is understood to be specifically contemplated and described herein even if not explicitly disclosed. Thus, if classes of molecules A, B, and C and classes of molecules D, E, and F are disclosed and, by way of example, combination molecule A-D is disclosed, they are considered individually and collectively even if not each individually described. Thus, in this example, each combination A-E, A-F, B-D, B-E, B-F, C-D, C-E, C-F is specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the exemplary combination A-D. Similarly, any subset or combination of these is specifically contemplated and disclosed. Thus, for example, sub-groups of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the exemplary combination A-D. This concept applies to all aspects of the present application, including but not limited to the steps in methods of making and using the disclosed compositions. Thus, if there are various additional steps that are possible, it is understood that each of these additional steps is possible with any particular embodiment or combination of embodiments of the disclosed methods and that each such combination is specifically contemplated and should be considered disclosed.

[0057] The headings are provided for convenience only and are not to be construed as limiting the invention in any way. Embodiments shown in any heading or any part of the present disclosure can be combined with embodiments shown in the same or other headings or other parts of the present disclosure.

[0058] A. Definitions It is understood that the methods and compositions disclosed are not limited to the specific methodologies, protocols, and reagents described, as these can vary. It is also understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is limited only by the appended claims.

[0059] Also, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the MGS peptide" includes a plurality of such MGS peptides, and reference to "the linker" is a reference to one or more linkers known to those of ordinary skill in the art and their equivalents.

[0060] As used herein, "treatment" means administering a composition of the invention to a subject, such as a human or other mammal (e.g., an animal model) having a disease or condition, in order to prevent or delay the worsening of the effects of the disease or condition, or to partially or completely reverse the effects of the disease or condition. In some embodiments, the disease or condition may be a CNS-related disease or condition, or a CNS disorder or injury. Treatment may be administered to a subject that does not exhibit symptoms of a disease, disorder, and / or condition, and / or to a subject that exhibits only initial symptoms of a disease, disorder, and / or condition, for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment includes transporting one or more of the disclosed compositions to the subject.

[0061] As used herein, "prevention" means minimizing the likelihood that a subject predisposed to developing a disease, disorder, or medical condition will develop the disease, disorder, or medical condition.

[0062] As used herein, the term "subject" refers to a subject for 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. Also, the term "subject" includes pets (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, flies, etc.). In one embodiment, the subject is a mammal. In another embodiment, the subject is a human. This term does not indicate a specific age or gender. Thus, it is intended to include fetuses, regardless of whether they are adults, children, adolescents, newborns, or 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 method, the "patient" has been diagnosed as in need of treatment prior to the administration step. In some embodiments, the patient and the subject may be used interchangeably.

[0064] As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, symbols, or words representing amino acid residues. The abbreviations of amino acids used herein are the conventional one-letter codes of 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 contiguous amino acids. The term "polypeptide" includes naturally occurring molecules or synthetic molecules.

[0066] Furthermore, as used herein, the term "polypeptide" refers to amino acids joined to each other by peptide bonds or modified peptide bonds such as peptide isosteres, and may include modified amino acids other than the 20 amino acids encoded by the genes. A polypeptide may be modified by any of natural processes such as post-translational processing or chemical modification techniques known in the art. Modifications can occur anywhere on the polypeptide, such as on the peptide backbone, amino acid side chains, amino terminus, or carboxyl terminus. The same type of modification may be present in the same or varying degrees at several sites on 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 phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of an amino acid to a protein via a transfer RNA such as arginylation (see 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, New York, 1983, pp. 1-12).

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

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

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

[0070] The term "percent(%) homology" is used interchangeably with the term "percent(%) identity" in this specification, 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 an alignment program. For example, as used herein, 80% homology means the same as 80% sequence identity determined by a defined algorithm, and thus, a homolog of a given sequence has a sequence identity greater than 80% over 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. Also see Altschul et al. in 1990 and Altschul et al. in 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 utilizing the BLOSUM-62 matrix (see, for example, Altschul, S.F., et al. Nucleic Acids Res. 25:3389-3402, 1997). For example, a preferred alignment of sequences selected to determine "% identity" between two or more sequences is performed using the CLUSTAL-W program of Mac Vector version 13.0.7, and is 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] To reach the final derivatives, variants, or analogs, substitutions, deletions, insertions, or any combination thereof may be used. Generally, these changes are made to a few nucleotides to minimize changes to the molecule. However, in some situations, larger changes may be tolerated.

[0072] Generally, the nucleotide identity between individual variant sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a "variant sequence" has a specific identity to the parental or reference sequence (e.g., wild-type sequence) of the present invention and shares a biological function that includes, but is not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parental sequence. For example, a "variant sequence" may be a sequence that contains one, two, or three, four nucleotide base changes compared to the parental or reference sequence of the present invention and shares or improves the biological function, specificity, and / or activity of the parental sequence. Thus, a "variant sequence" may have a specific identity to the parental sequence of the present invention and share a biological function that includes, but is not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parental sequence. Also, the variant sequence may share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the reference sequence (e.g., MTS sequence).

[0073] "Any" or "optionally" means that the event, situation, or material described thereafter may or may not occur or exist, and that both the case where the event, situation, or material occurs or exists and the case where it does not occur or exist are included in the description.

[0074] In this specification, a range may be expressed as "about" a particular value and / or "about" to another particular value. Also, when such a range is expressed, unless specifically indicated in the context, the range from one particular value and / or to another particular value is specifically considered and is deemed to be disclosed. Similarly, when a value is expressed as an approximate value, by using the antecedent "about", unless specifically indicated in the context, the particular value is understood to form another specific embodiment to be specifically considered as being disclosed. Further, it is understood that each endpoint of a range is significant both when related to other endpoints and when independent of other endpoints, unless specifically indicated in the context. Finally, it is understood that all individual values and sub-ranges of values included within an explicitly disclosed range are to be specifically considered and deemed to be disclosed, unless specifically indicated in the context. The above applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular case.

[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. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions, but the particularly useful methods, devices, and materials are as described. Publications cited herein and the materials they cite are specifically incorporated herein by reference. No admission is made that any description herein constitutes prior art to the present invention. No document is admitted to be prior art. The description of a reference is what its author has claimed, and the applicant reserves the right to challenge the accuracy and validity of the cited documents. Although many publications are referenced herein, it is to be clearly understood that such references do not admit that any of these documents form part of the common general knowledge in the art.

[0076] Throughout the description of the present specification and the claims, the terms "comprising" and variations such as "comprises" and "comprising" mean "including but not limited to", and are not intended to exclude, for example, other additives, components, 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 described (unless the step includes limiting terms such as "consisting of"), and each step is not intended to exclude, for example, other additives, components, integers, or steps not described in that step.

[0077] B. Molecular Induction System (MGS) Peptide Disclosed herein are MGS peptides. In some embodiments, the MGS peptides may selectively bind to specific cell tumors. 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 a neuronal targeting molecule-guided system (“MGS”) peptide with high affinity and specificity that enables efficient transport of therapeutic molecules into neurons. By this technique, peptide sequences that can be used for neuronal targeting can be screened quickly and efficiently. The neuronal targeting MGS peptide exhibits high affinity for neurons and can be internalized into neurons. Furthermore, the neuronal targeting MGS peptide showed selectivity in brain cells (e.g., astrocytes, microglia).

[0079] The neuronal targeting MGS peptide may be conjugated with small molecules, nucleic acids, and antibodies for therapeutic purposes. Furthermore, the diverse chemical properties of the peptide sequence enable modifications that enhance the sensitivity and specificity of neuronal targeting and combinations with other peptides for dual or multi-targeting (e.g., combination with a blood-brain barrier-penetrating peptide). These neuronal targeting peptides also have the ability to induce high neuronal targeting sensitivity and specificity. Therefore, this neuronal targeting peptide has the potential as an induction system for the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0080] Disclosed is an MGS peptide comprising an amino acid sequence of any of the sequences of SEQ ID NO: 1-8 shown in Table 1. [Table 1]

[0081] Table 1 shows only the monomeric MGS peptide sequences, but the MGS peptides may be used as multimers such as dimers and tetramers. In some embodiments, when "-2" is appended to the end of the peptide name, it refers to the dimeric MGS peptide. In some embodiments, when "-4" is appended to the end of the peptide name, it refers to the tetrameric MGS peptide. For example, MGS_NOE3_V1-2 refers to the dimeric version of the GFHNVYPYTWGGFSDLMADEI (SEQ ID NO:1) sequence. In some embodiments, MGS_NOE3_V1-4 refers to the tetrameric version of the GFHNVYPYTWGGFSDLMADEI (SEQ ID NO:1) sequence. In some embodiments, MGS_NOE3_V2-2 refers to the 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. Thus, the same is true for EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2), where 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 the 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, MGS2_V4-2 are dimers respectively, and MGS_Neuron1_V2-4, MGS_Neuron2_V1-4, MGS_Neuron2_V2-4, MGS2_V3-4, 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 with any of the sequences of SEQ ID NO: 1-8. In some embodiments, one or more MGS peptides have 100% identity in the active portion of the peptide, and the active portion is 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 with any of the MGS peptides occurs outside of the active portion.

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

[0084] In some embodiments, the MGS peptides disclosed herein may be chemically conjugated to another MGS peptide, 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 of sufficient length to separate one or more MGS peptides from the cargo so as to prevent steric interference between the 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 the above PEG or another linker. In some embodiments, the linker may link two or more MGS peptides. In some embodiments, the linker may be conjugated to the cargo. By way of example, the MGS peptide comprising a linker may have a structure as shown in Figure 3, where X is the MGS peptide and R is a reactive group that can be conjugated to the cargo or another linker.

[0085] In some embodiments, the linker may be any of those described herein. For example, the linker may be of any length that enables conjugation between the MGS peptide and 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 combinations thereof.

[0087] In some embodiments, examples of the structure of the MGS peptide are shown in FIGS. 1 and 2. FIG. 1 shows the structure of the MGS2_V4 peptide sequence: CH3CO-YAAWPASGAWT (SEQ ID NO:8). FIG. 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 peptide may be a dimer, 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 peptide may be a tetramer, 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 group may be, but is not limited to, carboxylic acid, acyl halide, sulfonyl halide, chloroformate, aldehyde, alkyne, (alkyne without acetylenic hydrogen), amide and imide, amine, phosphine, and pyridine, anhydride, azo, diazo, azide, hydrazine, and azide compounds, carbamate, epoxide, ester, sulfate ester, phosphate ester, ester, thiophosphate ester, and borate ester, halogenated organic compound, isocyanate and isothiocyanate, ketone, oxime, sulfide (organic).

[0091] C. Molecular Transport System (MTS) Peptide Transport of cargo across the blood-brain barrier provides a unique opportunity to affect the delivery of cargo (e.g., drugs and polymers) to the CNS. Accordingly, this specification discloses peptides for targeting the CNS, often referred to as molecular transport system (MTS) peptides.

[0092] This specification discloses MTS peptides. Disclosed are MTS peptides comprising an amino acid sequence of any of the sequences of SEQ ID NO:9-14 shown in Table 2. [Table 2]

[0093] Table 2 shows only monomeric MTS peptide sequences, but MTS peptides may be used as multimers such as dimers. In some embodiments, a peptide name ending with "-2" refers to a dimeric MTS peptide. For example, MTS1_V2-2 refers to a 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 optimization such as lipidation is added, it refers to the next version or "V" of that peptide. For example, MTS3_V2-2 is a 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 with 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, and the active portion is the portion that retains the ability to move from blood to CSF. Thus, in some embodiments, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with any of the MTS peptides occurs outside the active portion.

[0095] In some embodiments, the MTS peptide may be modified. In some embodiments, modification of the MTS peptide includes optimizing the peptide or stabilizing the peptide.

[0096] In some embodiments, the MTS peptide may be stabilized such that the MTS peptide remains intact (e.g., does not degrade) during synthesis and / or storage. In some embodiments, the MTS peptide is stabilized by changing glycine to alanine. In some embodiments, glycine at position 2 of SEQ ID NO: 9 or 10 can be stabilized by changing it to alanine.

[0097] In some embodiments, the MTS peptide may be optimized. The optimized peptide can be obtained by adding modifications to the individual parent peptide sequences. These modifications can be used to identify the essential amino acids within the parent sequence required for movement from blood to CSF. These modifications can be obtained by a combination of alanine scanning and truncation of the amino-terminal and C-terminal regions of the parent peptide. PEG12 can protect the C-terminus of the MTS peptide, provide a spacer between the peptide and the cargo molecule conjugated via the C-terminal cysteine, and enhance the solubility of the MTS peptide. Modification of the N-terminus of the MTS peptide with an acetyl group (CH3CO-) and / or a d-amino acid 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 may be tested to confirm their 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 can be anything that prevents proteases from shortening the amino acids from the N-terminus. In some embodiments, the MTS peptides disclosed herein may have their N-terminus 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 can be, but is not limited to, PEG, formyl, CH3-(CH)n-CO, fluorophore, fatty acid, alkylamine, sulfonamide, aryl group, carbohydrate, D-amino acid, or 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 of sufficient length 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 the 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 the above PEG or another linker. In some embodiments, the linker may link two or more MTS peptides. In some embodiments, the linker may be conjugated to the cargo. By way of example, an MTS peptide comprising a linker may have a structure as shown in Figure 3, where X is an MTS peptide and R is a reactive group that can be conjugated to the cargo or another linker.

[0100] In some embodiments, the MTS peptides disclosed herein may be shortened. In some aspects, the MTS peptides disclosed herein are shortened 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 aspects, the active portion can be determined using techniques known in the art, such as alanine scanning or truncation studies. The active portion of the MTS peptide is the portion that retains the ability to move from blood to CSF. For example, SKETYSMNAQRQHERS (SEQ ID NO:13) can be truncated at the N-terminus 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 embodiments, 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 the MTS peptide Ac-DAYKLQTSLDWQMWNP (SEQ ID NO:10), also referred to as MTS1_V2. Figure 4B shows an example of the MTS peptide Ac-FPSWTSKNQQWTNQRQ (SEQ ID NO:12), also referred to as MTS1_V2. Figure 4C shows an example of the MTS peptide Ac-SKETYSMNAQRQHERS (SEQ ID NO:14), also referred to as MTS3_V1.

[0103] In some embodiments, the disclosed MTS peptides may be dimers, and two MGS peptides may be conjugated. Figure 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 group may be, but is not limited to, carboxylic acid, acyl halide, sulfonyl halide, chloroformate, aldehyde, alkyne, (alkyne without acetylenic hydrogen), amide and imide, amine, phosphine, and pyridine, anhydride, azo, diazo, azide, hydrazine, and azide compounds, carbamate, epoxide, ester, sulfate ester, phosphate ester, ester, thiophosphate ester, and borate ester, halogenated organic compound, isocyanate and isothiocyanate, ketone, oxime, (organic) sulfide.

[0105] D. Linker A linker is disclosed. In some embodiments, the linker can conjugate or bind two or more MGS peptides, two or more MTS peptides, two or more linkers, or a linker or peptide and a cargo.

[0106] In some embodiments, a linker that conjugates two or more MGS peptides or two or more MTS peptides may also be referred to as a dimer core (when binding to two peptides) or a tetramer core (when binding to four peptides). Figure 3 shows examples of the structures of both the dimer core and the tetramer core. In Figure 3, the linker comprises PEG12, but in some embodiments, PEG of any length may be used. For example, any of PEG1 - PEG30 may be used. In some embodiments, PEG with a length of 1 - 5000 may be used. In some embodiments, any linker may be used at the position of PEG12 of the dimer core or tetramer core.

[0107] A linker is disclosed that comprises at least one reactive group capable of binding 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 up to PEG5000. 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 to 4 PEG linkers. In some embodiments, a linker comprising two PEG linkers may be referred to as a dimer core. In some embodiments, a linker comprising 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 at least two PEG linkers. In some embodiments, the reactive group connects at least two PEG linkers.

[0110] In some embodiments, the linker comprises an amino acid, a peptide, an alkyl group, maleimide, thiol, hydrazone, or amide. In some embodiments, the amino acid may be a modified amino acid. For example, the modified amino acid may 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] having the structure of JPEG0007684523000007.jpg23144, where X is a peptide and R is a reactive group that can react chemically with a moiety. In some embodiments, X may be an MGS peptide or an MTS peptide. In some embodiments, this structure may be referred to as a dimer core because it can bind two peptides.

[0112] In some embodiments, the linker may further comprise a lipid moiety. For example, one of the R groups from the dimer core may react with the lipid moiety. Thus, in some embodiments, the linker is [Formula 6] It has the structure of JPEG0007684523000008.jpg47144, where X is a peptide and R is a reactive group that can chemically react with a moiety. In some embodiments, X may be an MGS peptide or an MTS peptide. In some embodiments, X may be an MGS peptide or an MTS peptide.

[0113] In some embodiments, the linker is [Formula 7] It has the structure of JPEG0007684523000009.jpg60144, where X is a peptide and R is a reactive group that can chemically react with a moiety. In some embodiments, X may be an MGS peptide or an MTS peptide. In some embodiments, since this structure can bind four peptides, it may be referred to as a tetramer core. Another example of a tetramer core is [Formula 8] It has the structure of JPEG0007684523000010.jpg45144, where X is a peptide and R is a reactive group that can chemically react with a moiety. In some embodiments, X may be an MGS peptide or an MTS peptide. In some embodiments, the reactive group may be, but is not limited to, carboxylic acid, acyl halide, sulfonyl halide, chloroformate, aldehyde, alkyne, (alkyne without acetylenic hydrogen), amide and imide, amine, phosphine, and pyridine, anhydride, azo, diazo, azide, hydrazine, and azide compounds, carbamate, epoxide, ester, sulfate ester, phosphate ester, thiophosphate ester, and borate ester, halogenated organic compound, isocyanate and isothiocyanate, ketone, oxime, sulfide (organic).

[0114] In some embodiments, the moiety may be 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. Accordingly, in some embodiments, linkers comprising linkers are disclosed. For example, when the linker is a tetramer core (i.e., the 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 conjugates the tetramer core (i.e., the third linker) to a 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. Compositions Compositions comprising one or more of the disclosed MGS peptides, MTS peptides, linkers, and / or combinations thereof are disclosed.

[0117] Compositions comprising at least one MGS peptide conjugated to at least one MTS peptide are disclosed, wherein the MGS peptide comprises 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).

[0118] A composition is disclosed that comprises two or more MGS peptides conjugated to two or more MTS peptides, wherein the MGS peptides comprise 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).

[0119] In some embodiments, the two or more MGS peptides are four MGS peptides. Thus, for example, four MGS peptides conjugated to at least two MTS peptides comprise 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).

[0120] A composition is disclosed that comprises four MGS peptides conjugated to two MTS peptides, wherein the four MGS peptides comprise 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); and the two MTS peptides comprise the amino acid sequence 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, if 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, two or more MTS peptides are the same MTS peptide. For example, in some embodiments, there are two MTS peptides in the disclosed composition, and both of the MTS peptides are the same MTS peptide. In some embodiments, there are two MTS peptides in the disclosed composition, and the two MTS peptides are different from each other.

[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 binds 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, 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or a combination thereof. In some embodiments, the first linker may bind the MGS peptide to another MGS peptide, or bind the MGS peptide to a cargo, or bind 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 bind to a linker such as PEG (of any length). Next, each MGS peptide-PEG may be bound using one or more amino acids, modified amino acids, or linkers capable of creating the dimeric or tetrameric cores described herein.

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

[0126] In some embodiments, the third linker binds to the second linker. In some embodiments, the third linker binds 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 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. In some embodiments, the third linker may bind the MTS peptide to another MTS peptide, or bind the MTS peptide to a cargo, or bind the MTS peptide to another linker. In some embodiments, the third linker may comprise one or more of the structures shown in Figure 3. For example, each MTS peptide may be bound to a linker such as PEG (of any length). Next, each MTS peptide-PEG may be bound using one or more amino acids, modified amino acids, or linkers capable of creating the dimeric or tetrameric cores described herein.

[0127] In some embodiments, 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, 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 comprises a PEG linker. In some embodiments, a PEG linker of any length may be used. For example, any of PEG1-PEG30 may be used. In some embodiments, a PEG of length 1-5000 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 siRNA. In some embodiments, the cargo binds to a first linker, a second linker, and / or a third linker. Thus, in some embodiments, the disclosed composition may comprise one or more cargos. 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 an 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 anything that prevents proteases from shortening the 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 may 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. In some embodiments, the two MTS peptides are linked via the dimer core described.

[0131] 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), a first linker comprising (i) at least one reactive group capable of binding 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 comprising (i) at least one reactive group capable of binding to the C-terminus of the peptide and (ii) at least one additional reactive group capable of chemically reacting with a moiety, an MTS peptide comprising the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13), are provided in a composition.

[0132] In some embodiments, the C-terminus of the MGS peptide comprising the amino acid sequence of GFHNVYPYTWGGFSDIDLMADEI (SEQ ID NO:1); EQRWVQMLHLQTRYAGEWPG (SEQ ID NO:2); or FQHNPFPYTYSMEDTDVEIK (SEQ ID NO:3) binds to the first linker. In some embodiments, the first linker binds to the second linker. In some embodiments, the second linker binds to the third linker. In some embodiments, the third linker binds 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] Accordingly, in some embodiments, a composition is disclosed 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) that binds via a reactive group to a first linker, wherein the first linker binds to a second linker, the second linker binds to a third linker, and the third linker binds 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 [Formula 9] has a structure of JPEG0007684523000011.jpg98144, where X is an MGS peptide, X' is an MTS peptide, and R is a reactive group that can react chemically with a moiety. In some embodiments, the MGS peptide is any of those 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 of those 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 [Formula 10] It has the structure of JPEG0007684523000012.jpg62144, where X' is an MTS peptide, X is an MGS peptide, and R is a reactive group that can chemically react with a moiety. In some embodiments, the MGS peptide is any of those described herein. In some embodiments, the MGS peptide comprises 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 of those described herein. In some embodiments, the MTS peptide comprises 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 [Formula 11] It has the structure of JPEG0007684523000013.jpg41144, where X' is an MTS peptide, X is an MGS peptide, and R is a reactive group that can chemically react with a moiety. In some embodiments, the MGS peptide is any of those described herein. In some embodiments, the MGS peptide comprises 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 of those described herein. In some embodiments, the MTS peptide comprises an amino acid sequence of any of the sequences of SEQ ID NOs: 9-14 shown in Table 2.

[0137] In some embodiments, the reactive group may be, but is not limited to, carboxylic acid, acyl halide, sulfonyl halide, chloroformate, aldehyde, alkyne, (alkyne without acetylenic hydrogen), amide and imide, amine, phosphine, and pyridine, anhydride, azo, diazo, azide, hydrazine, and azide compound, carbamate, epoxide, ester, sulfate ester, phosphate ester, ester, thiophosphate ester, and borate ester, halogenated organic compound, isocyanate and isothiocyanate, ketone, oxime, sulfide (organic).

[0138] A pharmaceutical composition is disclosed. Accordingly, the disclosed composition further comprises a pharmaceutically acceptable carrier. A composition is disclosed that comprises an MGS peptide having an amino acid sequence of any one of the sequences of SEQ ID NO: 1-8 shown in Table 1 and a pharmaceutically acceptable carrier. A composition is disclosed that comprises an MTS peptide having an amino acid sequence of any one of the sequences of SEQ ID NO: 9-14 shown in Table 2 and a pharmaceutically acceptable carrier. A composition is disclosed that comprises four MGS peptides conjugated to two MTS peptides and a pharmaceutically acceptable carrier, wherein the four MGS peptides have an 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), and the two MTS peptides have an amino acid sequence 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 transport (or administration) of the peptides or compositions disclosed herein may occur via various mechanisms. As defined above, the present specification may be a composition comprising any one or more of the peptides described herein that may be used to create a composition that may also include carriers such as pharmaceutically acceptable carriers. For example, a pharmaceutical composition is disclosed that comprises a peptide disclosed herein and a pharmaceutically acceptable carrier.

[0140] For example, the compositions described herein may comprise a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material or carrier that, as is well known to those skilled in the art, will minimize any degradation of the active ingredient and will be selected to minimize side effects in the subject. Examples of carriers include dimyristoyl phosphatidylcholine (DMPC), phosphate buffered saline, or multilamellar liposomes. For example, PG:PC:cholesterol:peptide or PC:peptide may be used as carriers in the present invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in A.R. Gennaro, Remington: The Science and Practice of Pharmacy (19th edition), Mack Publishing Company, Easton, 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Other examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution may be from about 5 to about 8, or from about 7 to about 7.5. Further carriers are sustained release formulations such as the semipermeable matrix of a solid hydrophobic polymer containing the composition, which matrix is in the form of, for example, a film, a stent (implanted into a blood vessel during angioplasty), a liposome, or microparticles. For example, it will be apparent to those skilled in the art that certain carriers may be more preferred depending on the route of administration and the concentration of the composition being administered. Most typically, these are standard carriers for drug administration to humans, including solutions such as sterile water, saline, buffers at physiological pH, and the like.

[0141] Also, the pharmaceutical composition may include carriers, thickeners, diluents, buffers, preservatives, etc., as long as the intended activity of the polypeptides, peptides, nucleic acids, vectors of the present disclosure is not impaired. Further, the pharmaceutical composition may include one or more active ingredients such as antibacterial agents, anti-inflammatory agents, anesthetics, etc. (in addition to the compositions of the present disclosure). The pharmaceutical composition may be administered in many ways depending on whether local treatment or systemic treatment is desired and on the site being treated.

[0142] The preparation of parenteral formulations includes 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, alcohol / aqueous solutions, emulsions, or suspensions including physiological saline and buffer media. Parenteral vehicles include sodium chloride solution, Ringer's glucose, glucose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles are body fluids and nutrient supplements, electrolyte supplements (such as those based on Ringer's glucose), etc. For example, preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, and inert gases may also be present.

[0143] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, powders, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oil-based, thickeners, etc. are required 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, flavors, diluents, emulsifiers, dispersion aids, or binders are desirable. Some of the compositions may be administered as pharmaceutically 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, or formed by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl, arylamine, substituted ethanolamine.

[0145] The disclosed transport technology may be used for the disclosed compositions as well as for the disclosed nucleic acid sequences and vectors.

[0146] F. Method of transporting cargo A method of transporting cargo to the CNS of a subject, comprising administering to the subject in need thereof one or more of the compositions disclosed herein, wherein the peptide conjugated 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 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 enter the CNS across the blood-brain barrier. In some embodiments, the MGS peptide targets the cargo to specific CNS cells. In some embodiments, the CNS cells are neurons or microglia.

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

[0151] In some embodiments, the administration is intravenous or intrathecal. In some aspects, any of the transport methods described herein may be used.

[0152] In some embodiments, the composition is a composition comprising four MGS peptides conjugated to two MTS peptides, the four MGS peptides 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), and the two MTS peptides having the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0153] As described herein, the composition may further comprise one or more linkers. In some embodiments, the disclosed composition may further comprise a first linker. In some embodiments, the first linker binds 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 comprise a PEG linker, an alkyl linker, a maleimide linker, an amide linker, a peptide linker, an amide linker, an aryl linker, a dibenzoctyne linker, an azide linker, a triazole linker, a dibenzoazacyclooctyne linker, 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or combinations thereof. In some embodiments, the first linker may bind an MGS peptide to another MGS peptide, or an MGS peptide to a cargo, or an 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 bound to a linker such as PEG (of any length). Next, each MGS peptide-PEG may be bound using one or more amino acids, modified amino acids, or linkers capable of creating the dimeric or tetrameric cores described herein.

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

[0155] In some embodiments, the third linker binds to the second linker. In some embodiments, the third linker binds 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 comprise a PEG linker, an alkyl linker, a maleimide linker, an amide linker, an aryl linker, a peptide linker, a dibenzoctyne 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. In some embodiments, the third linker may bind an MTS peptide to another MTS peptide, or an MTS peptide to a cargo, or an MTS peptide to another linker. In some embodiments, the third linker may comprise one or more of the structures shown in Figure 3. For example, each MTS peptide may bind to a linker such as PEG (of any length). Next, each MTS peptide-PEG may be bound using one or more amino acids, modified amino acids, or linkers capable of creating the dimeric or tetrameric cores described herein.

[0156] In some embodiments, 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 dibenzoctyne 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. In some embodiments, the first, second, and / or third linker comprises a PEG linker. In some embodiments, linkers of any length may be used. For example, any of PEG1 to PEG30 may be used. In some embodiments, PEGs of lengths from 1 to 5000 may be used.

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

[0158] In some embodiments, the MGS peptide, the MTS peptide, or both have their N-termini protected. For example, in some embodiments, the MGS peptide, the MTS peptide, or both have their N-termini acetylated. Thus, in some embodiments, the MGS peptide or the MTS peptide may be any of those in Table 1 and / or Table 2.

[0159] G. Treatment Methods Disclosed is a method of treating a CNS disorder or injury in a subject in need thereof, comprising administering to the subject 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), gene therapy agent, compound, nucleic acid sequence, small molecule, ribonucleoprotein, or peptide (or protein). In some embodiments, specific examples of CNS disorder or injury therapeutic agents may include, but are not limited to, N-methyl D-aspartic acid (NMDA) antagonists, chemotherapeutic agents, glutamate antagonists, or immunomodulatory agents (e.g., immunosuppressive agents or immunostimulatory agents). In some embodiments, gene therapy enables the transport of genetic material encoding a therapeutic molecule. In some embodiments, gene therapy involves the administration of a biological pharmaceutical containing a recombinant nucleic acid that is administered to a subject to regulate, repair, replace, add, or delete a gene sequence for the purpose of treating or curing a disease. In some embodiments, the nucleic acid may be, but is not limited to, small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs). In some embodiments, the therapeutic agent may be an antibody targeting Aβ peptide / plaque (such as Donanemab, Lecanemab), tau tangles, β-secretase, γ-secretase, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE). In some embodiments, nucleic acid therapy that reduces the expression of Aβ peptide, tau tangles, β-secretase, γ-secretase, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), CD22 may be used

[0161] In some embodiments, the composition is a composition comprising four MGS peptides conjugated to two MTS peptides, the four MGS peptides 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), and the two MTS peptides having the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0162] As described herein, the composition may further comprise one or more linkers. In some embodiments, the disclosed composition may further comprise a first linker. In some embodiments, the first linker binds 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 comprise 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-dibenzotriazolo[4,45-d]azocine, or combinations thereof. In some embodiments, the first linker may bind an MGS peptide to another MGS peptide, or an MGS peptide to a cargo, or an 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 bound to a linker such as PEG (of any length). Next, each MGS peptide-PEG may be bound using one or more amino acids, modified amino acids, or linkers capable of creating the dimeric or tetrameric cores described herein.

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

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

[0165] In some embodiments, the third linker binds 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 may comprise 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. In some embodiments, the third linker may bind an MTS peptide to another MTS peptide, or an MTS peptide to a cargo, or an MTS peptide to another linker. In some embodiments, the third linker may comprise one or more of the structures shown in Figure 3. For example, each MTS peptide may bind to a linker such as PEG (of any length). Next, each MTS peptide-PEG may be bound using one or more amino acids, modified amino acids, or linkers capable of creating the dimeric or tetrameric cores described herein.

[0166] In some embodiments, 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 dibenzoctyne 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. In some embodiments, the first, second, and / or third linker comprises a PEG linker. In some embodiments, linkers of any length may be used. For example, any of PEG1 to PEG30 may be used. In some embodiments, PEGs having a length of 1 to 5000 may be used.

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

[0168] In some embodiments, the MGS peptide, the MTS peptide, or both have their N-terminus protected. For example, in some embodiments, the MGS peptide, the MTS peptide, or both have their N-terminus acetylated. Thus, in some embodiments, the MGS peptide or the MTS peptide may be any of those in Table 1 and / or Table 2.

[0169] H. Imaging Method Disclosed is a method of imaging the CNS of a subject comprising administering to the subject in need thereof one or more of the compositions disclosed herein, 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 binds to a protein, a peptide, or a nucleic acid.

[0170] In some embodiments, the composition is a composition comprising four MGS peptides conjugated to two MTS peptides, the four MGS peptides 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), and the two MTS peptides having the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO:9); FPSWTSKNQQWTNQRQ (SEQ ID NO:11); or SKETYSMNAQRQHERS (SEQ ID NO:13).

[0171] As described herein, the composition may further comprise one or more linkers. In some embodiments, the disclosed composition may further comprise a first linker. In some embodiments, the first linker binds 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 comprise 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-dibenzotriazolo[4,5-d]azocine, or combinations thereof. In some embodiments, the first linker may bind an MGS peptide to another MGS peptide, or an MGS peptide to a cargo, or an 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 bound to a linker such as PEG (of any length). Next, each MGS peptide-PEG may be bound using one or more amino acids, modified amino acids, or linkers capable of creating the dimeric or tetrameric cores described herein.

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

[0173] In some embodiments, the third linker binds to the second linker. In some embodiments, the third linker binds 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 comprise 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, 8,9-dihydro-1H-dibenzo[1,2,3]triazolo[4,45-d]azocine, or combinations thereof. In some embodiments, the third linker may bind an MTS peptide to another MTS peptide, or an MTS peptide to a cargo, or an MTS peptide to another linker. In some embodiments, the third linker may comprise one or more of the structures shown in Figure 3. For example, each MTS peptide may bind to a linker such as PEG (of any length). Next, each MTS peptide-PEG may be bound using one or more amino acids, modified amino acids, or linkers capable of creating the dimeric or tetrameric cores 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 to which PEG can be attached, resulting in a dimer. In the case of a tetramer, a fully deprotected lysine may be bound to another lysine during peptide synthesis. As a result, four free amino groups can be obtained for use in attaching PEG.

[0174] In some embodiments, 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 dibenzoctyne 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. In some embodiments, the first, second, and / or third linker comprises a PEG linker. In some embodiments, linkers of any length may be used. For example, any of PEG1-PEG30 may be used. In some embodiments, PEGs of length 1-5000 may be used.

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

[0176] In some embodiments, the MGS peptide, the MTS peptide, or both have their N-terminus protected. For example, in some embodiments, the MGS peptide, the MTS peptide, or both have their N-terminus acetylated. Thus, in some embodiments, the MGS peptide or the MTS peptide may be any of those in Table 1 and / or Table 2.

[0177] I. Dosage A dosing regimen is disclosed that comprises administering one or more of the disclosed compositions or peptides in a single dose to a subject in need thereof, the single dose comprising an amount effective to enter the CNS and target a specific CNS cell type.

[0178] There is disclosed an administration 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 at the same concentration. In some embodiments, each dose after the first dose may be reduced. In some embodiments, each dose after the first dose may be increased.

[0179] In some embodiments, a single dose may be a continuous administration. In some embodiments, the continuous administration may be for several hours, several days, several weeks, or several months. In some embodiments, there may be two or more doses. In some embodiments, the two or more doses may be administered at several days, several weeks, or several months intervals.

[0180] J. Kits The above materials and other materials can be packaged together in any suitable combination as kits useful for performing or assisting in the performance of the disclosed methods. A kit is useful if the kit components given are designed and adapted to be used together in the disclosed methods. For example, there is disclosed a kit comprising one or more of the disclosed MGS peptides, MTS peptides, linkers, or combinations thereof. For example, there is disclosed a kit comprising any of the disclosed compositions.

[0181] A. Example 1: Dual Targeting Figure 5 shows an example of the structure of a dimer core modified with a fatty acid. 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] Figure 6 shows a schematic diagram of a general experimental flow used to evaluate the transport of the disclosed compositions to the CNS.

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

[0184] Brain accumulation was improved with MTS3_V2-2 compared to the parental MTS3_V1-2, as demonstrated by brain sectioning and fluorescence imaging (Figure 8). Coronal slices were 1 mm and were arranged from anterior (upper left) to posterior (lower right). Consistent with the data from brain homogenates, MTS3_V2-2 (C16 fatty acid modification) showed increased brain uptake compared to MTS3_V1-2 at all time points. Fluorescent signals were seen throughout the brain, indicating diffusion within the parenchyma.

[0185] Figure 3 shows an example of the core structure used in the microglia targeting experiment described below. Figure 3A shows a dimer core, and Figure 3B shows a tetramer core. The shown core structures comprised the MGS peptide represented as X. The specific MGS peptide used in the following experiment was MGS2_V4, CH3CO-YAAWPASGAWT. Specifically, the tetramer core MGS2_V4-4 was used. CH3O-SS

[0186] MGS2_V4-2 was specific to microglial cells and promoted high-level cell uptake (Figure 9). Figure 9A shows that MGS2_V4-2 bound to and internalized into microglial cells with high affinity, and Figure 9B shows that MGS2_V4-2 bound to and internalized into 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 the 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 minutes was observed at 1 hour and continued to increase over time. The peaks were integrated to determine the proportion of the inactive peptide and the amount of the observed degradation product. After 24 hours, more than 75% of MGS2_V4-2 remained inactive. Electrospray mass spectrometry was performed on the peptide product that occurred at a retention time of 10.005 minutes. 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 in the CNS. The graph in Figure 12 shows examples of the uptake of MGS2_V4-2, MGS3_V1-2-MGS2_V4-2 chimeras, 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 incubation at 37 °C for 1 hour, uptake was measured by quantitative flow cytometry. MGS2_V4-2 and MTS3_V1-2-MGS2_V4-2 chimeras showed the same EC50 and intracellular uptake, indicating that the binding of MTS3_V1-2 did 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 rather than MTS.

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

[0190] Figure 14 shows the dual targeting of the MTS3_V1-2-MGS2_V4-2 chimera to microglia in the CNS. Staining was observed only in microglia. Microglial staining was not observed with MTS only, MGS only, or the scrambled control version of the conjugate.

[0191] Figure 15 shows the dual targeting of the MTS3_V1-2-MGS2_V4-2 chimera to microglia in the CNS as demonstrated by flow cytometry. The MTS3_V1-2-MGS2_V4-2 conjugate was labeled with Alexa Fluor 647 (red) and injected into the lateral tail vein of Sprague Dawley rats (0.09 nmol / g). After a 2-hour circulation time, the animals were euthanized and brain tissue was harvested. The brain was 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 remaining part of the brain using a Miltenyi QuadroMACS Separator. Thereafter, microglia were stained with an 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; Figure 15).

[0192] Figure 16 shows the structure of the MTS3_V1-2-MGS2_V4-4 chimera used in the siRNA transport experiment 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 the MGS2-MTS3 chimera in microglia is shown in Fig. 18. Fig. 18A shows the structure in which the chimeric MGS2_V4-4 (tetramer)-MTS3_V1-2 was conjugated to the test siRNA using click chemistry. Surprisingly, when siRNA was bound to MGS2_V4-2 (dimer)-MTS3_V1-2, the binding to HMC3 cells was lost. However, when the tetrameric MGS2_V4-4 was used as the targeting peptide of this multifunctional chimera, the binding was restored (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 tetrameric MGS_NOE3_V2-4 used in Figs. 20 to 24 showing neuronal targeting.

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

[0196] Figure 21 shows the tetramer MGS_NOE3_V2-4 that is specific for neurons and promotes high-level cell uptake. Monomeric and dimeric MGS_NOE3_V2-4 do not bind. To act, it must be a tetrameric core. Figure 21A shows the uptake of MGS_NOE3_V1-4, and MGS_NOE3_V2-4 was incubated with the indicated cell lines at 20 nM for 1 hour. The number of molecules of internalized peptide 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 prefrontal cortex), HMC3 (human microglial cell line), H1299, and H640 (both human non-small cell lung cancer cells). MGS_NOE3_V1-4 and MGS_NOE3_V2-4 showed 5- to 6-fold specificity for neurons over astrocytes and 3-fold specificity compared to microglia. Figure 21B shows that the uptake of MGS_NOE3_V2-4 into HT22 increased with increasing concentration. Uptake was also evaluated for the monomeric (MGS_NOE3_V2-1) and dimeric (MGS_NOE3_V2-2) versions, and no uptake was seen in HT22 cells, indicating that a tetrameric core is required to induce binding and uptake into neurons. Figure 21C shows that the 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 a flow cytometry assay.

[0197] Recycled the cell receptor of MGS_NOE3_V2-4 (Figure 22). HT-22 cells were incubated with a culture solution containing 100 nM MGS_NOE3_V2-4 in the presence of 100 μM chloroquine or 50 μM cycloheximide for the indicated time. Cell 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 continuous uptake. However, chloroquine, which inhibits endosomal acidification and transport, significantly decreased uptake from 1 hour of incubation and continued to suppress the internalization of MGS until 24 hours. Endosomal acidification was required for the continuous uptake of MGS_NOE3_V2-4. The uptake of MGS_NOE3_V2-4 continued over time. Collectively, these data indicate that the intracellular receptor of MGS_NOE3_V2-4 internalized, then returned to the cell surface, where it repeated the internalization of more MGS_NOE3_V2-4.

[0198] MGS_NOE3_V2-4 was internalized into neuronal cells (Figure 23). HT22 cells were incubated with 200 nM MGS_NOE3_V2-4-streptavidin-AlexaFluor647 conjugate at 37 °C for the indicated time. AlexaFluor488-conjugated wheat germ agglutinin was used for cell membrane labeling, and Hoechst 33342 was used for nuclear staining. Live cell imaging was performed, and representative images are shown. Intracellular red punctate staining was observed at 4 hours (Figure 23B) and continued to increase over time (Figure 23C). A control sample containing streptavidin 647 but not MGS_NOE3_V2-4 is shown in panel A. The absence of red staining indicates that MGS_NOE3_V2-4 mediated the uptake into cells. The red staining (green staining) located around the cells indicates the internalization of MGS_NOE3_V2-4 into cells.

[0199] Figures 24A through 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 circulating the peptide for 1 hour, the animals were euthanized and brain tissue was harvested. The whole brain was 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] Figure 35 shows the MTS3_V1-2-MGS_NOE3_V2-4 chimera used in the experiments shown in Figures 26 and 27.

[0201] Figures 26A through 26C show that the MTS3_V1-2-MGS_NOE3_V2-4 chimera was internalized into HT22 neurons. Intracellular punctate staining was observed at 1 hour and continued to increase over time. Figure 26A shows a control sample containing Alexa Fluor 647 but not MGS_NOE3_V2-4 and HT22 cells alone. The dual-targeted chimera MTS3_V1-2-MGS_NOE3_V2-4 was internalized into HT22 cells, as can be seen by the staining within the boundaries of the cell membrane (lower right in Figure 26C). Uptake of the dual-targeted 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-targeted chimera MTS3_V1-2-MGS_NOE3_V2-4 continued over time and reached approximately 250,000 molecules / cell (≈250 nM) at 24 hours. Collectively, these data show that the MTS3_V1-2-MGS_NOE3_V2-4 chimera retains the ability to be taken up into neurons, reaches intracellular levels of ≈250 nM after 24 hours, and that this uptake is driven by the MGS_NOE3_V2-4 component of the chimera.

[0202] Figure 27 shows that MTS2_V1-2 binds to the transferrin receptor and accesses the CNS, while MTS3_V1-2 utilizes a novel and yet unidentified cellular receptor for transport. Recombinant human transferrin receptor was adsorbed onto a high-binding ELISA plate at 1 μg / mL. Biotinylated MTS2_V1-2 or MTS3_V1-2 was incubated at various concentrations for 1 hour, after which the samples were removed and the plates were washed to remove excess peptide. The retained MTS peptide was detected using streptavidin-HRP and TMB reagent. 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 and saturated at higher concentrations. In comparison, MTS3_V1-2 and the scrambled control peptide of MTS2_V1-2 did not show 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 kon and koff rates were measured at various concentrations of human or mouse transferrin receptor, and the dissociation constant was determined. 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. Collectively, these data support that the transferrin receptor was the target of MTS2_V1-2. The receptor for MTS3_V1-2 remains unknown, but the data indicate that it is not the transferrin receptor, and this peptide is likely accessing the CNS by a mechanism different from MTS2_V1-2.

[0203] 1. Materials and Methods i. General methods for quantifying peptide internalization in cells Cells were incubated in complete medium at 37 °C with the desired peptide (MGS, MTS, or MGS-MTS conjugate) labeled with 3 Alexa Fluor 647. The peptide concentration, incubation time, and cell line used are shown in each figure. The peptide was removed, and the 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 using a BD FACSCelesta, and the data were analyzed using 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. For absolute peptide uptake per cell, Quantum TM A standard curve was generated using Quantum Alexa Fluor 647 microspheres. The MFI was determined at 50% of the peak height. The internalized molecules per cell were determined by a standard curve correlating MESF and MFI and divided by the number of dye molecules / MGS conjugate. GraphPad Prism® was used for non-linear regression curve fitting to calculate the EC50.

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

[0205] ii. CNS cell lines used in uptake studies All cell lines were maintained according to the manufacturer's protocol and medium supply.

Table 3

[0206] iii. In Vivo MTS and MTS-MGS Targeting Experiments An MTS peptide directly conjugated to the Alexa Fluor 750 dye (1 μg / g body weight) was injected into Sprague-Dawley rats via the lateral tail vein and circulated for the indicated times (20 minutes, 1 hour, 6 hours, 24 hours). Ten minutes before the end of the time period, the animals were anesthetized and 2 mL of 200-fold heparinized saline was injected intraperitoneally. To collect CSF, the back of the neck was bluntly dissected to expose the transparent dura mater covering the cerebral cistern. Using a glass micropipette, CSF was aspirated from the cerebral cistern at the indicated time points. After CSF collection, the heart was perfused with 500 mL of ice-cold 1xPBS containing heparin to remove the blood in the body. The brain was separated from other tissues. The brain was 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 generated using the MTS peptide conjugated to Alexa Fluor 750, and the amount of dye in the cerebrospinal fluid and brain was measured.

[0207] When using the brain for tissue sectioning, following the initial perfusion, perfusion is performed with 500 mL of ice-cold 5% paraformaldehyde. Coronal sections with a thickness of 1 mm were prepared using a brain matrix. These slices were imaged with an Odyssey Imager, and the fluorescence signal at 800 nm was collected.

[0208] iv. Confocal Microscopy: In Vitro Cell Analysis Test cells were seeded in 35 mm dishes for confocal microscopy 24 hours before treatment. Test peptides labeled with Alexa Fluor 647 were incubated on the cells at 37 °C for the time supported in the medium. After incubation, the 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 for cell membrane labeling, and Hoechst 33342 was used for nuclear staining. Microscopy was performed using a Zeiss LSM 700 equipped with a Pln Apo 63x / 1.4 oil DIC III objective lens. Images were processed using Zen software.

[0209] For biotinylated peptides instead of direct labeling with Alexa Fluor 647, the peptide - SA - AF647 conjugate was prepared by mixing Alexa FluorTM 647 conjugated streptavidin and biotinylated peptide in a 1:1 molar ratio in 100 μl of PBS at room temperature for 30 minutes. After conjugation, 900 μl of culture medium containing biotin (600 nM) was added to the mixture to saturate all binding sites on streptavidin. Then, the cell culture medium was exchanged with the mixture containing the peptide. The final peptide - SA - AF647 is shown in each figure. Microscopic observation was performed as described above.

[0210] v. Confocal microscopy and flow cytometry analysis for in - vivo transport experiments As described above, MTS, MGS, or MTS - MGS labeled with Alexa Fluor 647 were injected into Sprague - Dawley rats. After a 2 - hour circulation time, the animals were euthanized, and the brains were fixed by transcardial perfusion, harvested, cryoprotected, and cryosectioned into 15 - μm sections using a Leica CM1950. Images were taken using a Zeiss LSM 800. Microglia were stained with Iba - 1, and cell nuclei were stained with Hoechst 33342.

[0211] For the flow cytometry assay, the brain was dissociated using a Miltenyi Biotec gentleMACS Octo Dissociator. All 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 the Iba1 microglia marker. Flow cytometry data were collected using a BD FACSCelesta.

[0212] Alternatively, the brain was dissociated using a Miltenyi Biotec gentleMACS Octo Dissociator and all CNS cells were analyzed as a mixture. No enrichment of 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 the peptide conjugated to the dye (Alexa Fluor 647) was determined by absorbance at 651 nm and ~14 nmol of the peptide was lyophilized. The lyophilized peptide powder was dissolved in 400 μL of human serum (Innovative Research, H6430) to make a 35 μM solution. The samples were incubated at 37 °C with shaking. At each time point (0, 1, 2, 4, 20, 24 h), 50 μL aliquots were removed from the reaction and 100 μL of absolute ethanol was added to precipitate serum proteins. The samples were incubated on ice for 15 min and then centrifuged at 12,000 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 methods Figures 28 to 32 show examples of different reaction methods for creating 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 in a 45 mL reaction vessel and transferred to a peptide synthesizer (PurePep Chorus, Gyros Protein Technology) for automated synthesis. The resin was swollen in a 1:1 mixture of 6 mL of N,N-dimethylformamide (DMF, Fisher) / dichloromethane (DCM, Fisher) for 30 minutes, and then the moisture 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 in a 45 mL reaction vessel and transferred to a peptide synthesizer (PurePep Chorus, Gyros Protein Technology) for automated synthesis. The resin was swollen in a 1:1 mixture of 6 mL of N,N-dimethylformamide (DMF, Fisher) / dichloromethane (DCM, Fisher) for 30 minutes, and then the moisture was removed.

[0217] Manual addition of modified amino acids: The manual coupling cocktail (for the resin) generally contained 4.4 eq of HCTU (Gyros Protein Technology), 10 eq of N-methylmorpholine (NMM, Gyros Protein Technology), 5 eq of Oxyma pure (Gyros Protein Technology), and 5 eq of 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 of DMF. After 1 hour of coupling, the peptide resin was washed twice with 3 mL of DMF, twice with 3 mL of DCM, and once with 3 mL of DMF, and mixed for 20 seconds each time.

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

[0219] Manual addition of maleimide propionic acid: It contained 16 eq of N,N′-diisopropylcarbodiimide (DIC-Gyros Protein Technology), 16 eq of N-methylmorpholine (NMM, Gyros Protein Technology), 8 eq of 3-maleimide-propionic acid (Polypure), and 8 eq of Oxyma pure (Gyros Protein Technology) dissolved in 1 mL of DMF. After 2 hours of coupling, the peptide resin was washed again (as described 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 consecutive treatments (5 minutes and 10 minutes) with 2 mL of 20% piperidine (Sigma-Aldrich) in a 0.1 M Oxyma pure (Gyros Protein Technology) / DMF solution. After deprotection, washing was performed (as described above).

[0222] Coupling: The coupling step 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) in DMF, and 0.5 mL of 1 M N-methylmorpholine (NMM, Gyros Protein Technology) in DMF. After 30 minutes and 1 hour of consecutive coupling, the peptide resin was washed again (as described 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 consisted of two 20-minute treatments with a 2 mL solution of 0.2 mL acetic anhydride (Fisher), 0.2 mL NMM, and 1.6 mL DMF. After acetylation, it was washed 4 times with 3 mL of DMF and 8 times with 3 mL of DCM. Subsequently, the peptide resin was vacuum dried on a Prelude for 1 hour.

[0224] Cleavage: The peptide resin was transferred to a 5 mL filter syringe and cleaved with a 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 hours, poured into cold diethyl ether (Fisher), shaken vigorously to precipitate the peptide. The mixture was centrifuged at 2000 rpm for 2 minutes 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] Preparation and purification of crude peptides by RP-HPLC: The crude peptides were 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 hour. After dissolution, the peptide solution was filtered through a 0.2 mm filter syringe in preparation for RP-HPLC and analysis. The purification of the peptides 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 the mobile phase. Each injection was carried out using a Phenomenex Jupiter 5 mm C4 300A column (200x212 mm) at room temperature with a flow rate of 10 mL / min using a 20 - 70% Buffer B profile (20% from 0 - 3 minutes, 20 - 70% from 3 - 25 minutes, 90% from 25 - 30 minutes, 20% from 30 - 35 minutes). Fractions were collected, analyzed by analytical RP-HPLC and ESI-MS to evaluate purity, and lyophilized.

[0226] Conjugation of maleimide core and peptide (MGS or MTS): The lyophilized maleimide core was weighed and dissolved in 3 M GuHCl / PBS. Subsequently, the lyophilized MGS / MTS peptide was separately dissolved in 3 M GuHCl / PBS, the pH of the solution was determined, and adjusted to ~7 if necessary. These solutions were combined such that the maleimide core and the peptide were at a molar ratio of 1.0:2.2 eq. The reaction product was purified by preparative RP-HPLC (Agilent 1260) and then stirred for 3 hours. The peptide product was recovered, characterized by analytical RP-HPLC and ESI-MS, and lyophilized (Figure 29).

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

[0228] Synthesis of chimeric peptide: The lyophilized peptide conjugated with DBCO (MGS or MTS) and the peptide with Nα-Fmoc-Nε-azido-L-lysine incorporated into the core (MGS or MTS) were weighed and separately dissolved in 3 M GuHCl / PBS. The pH of each solution was determined and adjusted to ~7 if necessary. These solutions were combined such that the peptide conjugated with DBCO and the peptide conjugated with Nα-Fmoc-Nε-azido-L-lysine were at a molar ratio of 1.0:2.2 eq. After stirring the reaction overnight, it was purified by preparative RP-HPLC (Agilent 1260). The peptide product was recovered, characterized by analytical RP-HPLC and ESI-MS, and lyophilized (Figure 31).

[0229] Conjugation of siRNA and chimeric peptide: A chimeric peptide (composed of any core structure) having a free DBCO was dissolved in nuclease-free PBS (NF-PBS), and the pH was determined and adjusted to ~7 if necessary. Then, siRNA incorporating an azide at either the 5'-end or 3'-end was dissolved in NF-PBS, and the absorbance was measured at 260 nm to determine the concentration. These solutions were combined such that the siRNA and peptide were at a 1:1.2 molar eq. The reaction mixture was stirred for 3 to 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] One of ordinary skill in the art can recognize or ascertain many equivalents to the specific embodiments of the methods and compositions described herein without undue experimentation. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A peptide comprising the amino acid sequence of GFHNVPYTYWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWLVQMLHLQTRYAGEWPG (SEQ ID NO: 2); or FQHNPFPYTYSMEDTDEIK (SEQ ID NO: 3).

2. The peptide according to claim 1, which has an N-terminal protecting group.

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

4. The peptide according to any one of claims 1 to 3, further comprising a polymer linker or a chemical linker.

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

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

7. A molecular guiding system (MGS) peptide comprising the amino acid sequence of GFHNVPYTYWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWLVQMLHLQTRYAGEWPG (SEQ ID NO: 2); or FQHNPFPYTYSMEDTDEIK (SEQ ID NO: 3); a first linker comprising at least one reactive group capable of binding to the C-terminus of the MGS peptide and at least one additional reactive group capable of chemically reacting with a moiety; a second linker; a third linker comprising at least one reactive group capable of binding to the C-terminus of the MGS peptide and at least one additional reactive group capable of chemically reacting with another moiety; the MGS peptide comprising the amino acid sequence of DAYKLQTSLDWQMWNP (SEQ ID NO: 9); FPSWTSKNQQWTNQRQ (SEQ ID NO: 11); or SKETYSMNAQRQHERS (SEQ ID NO: 13); comprising, wherein the moiety and the other moiety comprise a composition containing a cargo.

8. The C-terminus of the MGS peptide comprising the amino acid sequence of GFHNVPYTYWGGFSDIDLMADEI (SEQ ID NO: 1); EQRWVMQLHLQTRYAGEWPG (SEQ ID NO: 2); or FQHNPFPYTYSMEDTDEIK (SEQ ID NO: 3) is bound to the first linker, the composition of claim 7.

9. The composition of claim 7 or 8, wherein the first linker is bound to the second linker.

10. The composition of claim 7 or 8, wherein the cargo comprises 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, a nanoparticle, or a linker comprising the second linker or another linker.

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