Molecular guide system peptides and uses thereof

Abstract

To provide a cell-targeting system that delivers a therapeutic nucleic acid to a specific cell type and to a specific location within the cell.SOLUTION: A composition comprising a nucleic acid sequence conjugated to one or more molecular guidance system (MGS) peptides.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 908, filed October 1, 2019. 687, which application is incorporated herein by reference in its entirety. There are.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made under Contract No. W81 awarded by the United States Medical Research Acquisition Agency (USAMRAA). Funding under XWH-16-1-0262 and by the National Institutes of Health Government support under grant numbers 7R01CA164447 and 7R01EB014244 This invention was made with support from the U.S. Government.

[0003] Sequence Listing Reference Created on July 30, 2020, "37794_0094P1_Sequence_ A text file named "Listing.txt" with a size of 18,505 bytes As filed, the sequence listing submitted on July 31, 2020, is in accordance with 37 CFR 1 .52(e)(5), which is incorporated herein by reference. [Background technology]

[0004] Cell targeting systems that deliver therapeutic nucleic acids to specific cell types and to specific locations within those cells. The system is needed.

[0005] The ability to deliver therapeutic agents to specific target cells and to the correct compartments within those cells has been demonstrated by many companies. Combining molecular guide systems (MGS) can have multiple health benefits. This will enable a new class of safe, targeted therapeutics with the ability to treat previously "undruggable" targets. This will enable the development of effective intracellular therapeutic drugs that can be used to treat emerging infectious diseases and viral infections. Treatment of neuroterrorism, cancer, diabetes, nervous system and neurodegenerative diseases, and genetic inheritance This opens up new therapeutic avenues for new treatments for diseases. Therapeutic drugs affect virtually every medical condition and represent a major breakthrough in modern medicine. This can create new products and create entirely new markets. Summary of the Invention

[0006] Disclosed are compositions comprising a nucleic acid sequence conjugated to one or more MGS peptides.

[0007] administering to the cells a composition comprising a nucleic acid sequence conjugated to one or more MGS peptides; 1. A method for modifying gene expression of a gene of interest, comprising: RNA transcribed from the target gene, the target gene, or the target The method further discloses a method for binding to a sequence upstream of a gene that is a target of the nucleotide sequence.

[0008] administering to the cells a composition comprising a nucleic acid sequence conjugated to one or more MGS peptides; a method for targeting a gene of interest, comprising: targeting an intracellular target, wherein the nucleic acid sequence is transcribed from the gene of interest within the intracellular target; The method further comprises binding to an RNA that is cleaved by the cleavage.

[0009] A method of treating a subject in need thereof, comprising administering to the subject in need thereof an effective amount of: administering a nucleic acid sequence conjugated to one or more MGS peptides, The method is disclosed, wherein the GS peptide targets an intracellular target involved in the disease process. .

[0010] Further advantages of the disclosed methods and compositions are described in part in the description that follows and are set forth in part in the specification. It may be understood in part from or learned by practicing the disclosed methods and compositions. The advantages of the disclosed methods and compositions are set forth with particularity in the appended claims. The foregoing general description and the following elements and combinations will be appreciated and achieved. The following detailed description is exemplary and explanatory only and does not limit the invention, as both are claimed. It should be understood that this is not a

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate the disclosed methods and Several embodiments of the method and composition are presented, and together with the description, the principles of the disclosed method and composition are demonstrated. It plays an explanatory role. [Brief explanation of the drawings]

[0012] [Figure 1] Schematic diagram of the molecular guidance system (MGS). Molecular targeting peptides are identified using FOX-Three technology. The peptides deliver cargo to target cells while avoiding uptake by other cells. Upon internalization, the peptides direct the cargo to a desired location within the cell. [Figure 2] FIG. 1 shows examples of cellular processes that can be manipulated by targeting therapeutic nucleic acids to specific intracellular organelles. [Figure 3]Figure 1 shows an example of MGS in which a fluorophore-labeled nucleic acid sequence card was internalized into four different cell types at two different temperatures, compared to the internalization profile of the fluorophore-labeled nucleic acid sequence without the aid of MGS. TmCAGmCATTmCTAATAGmCAGmC-Cy3 (SEQ ID NO: X) is the nucleic acid sequence used, conjugated to either hexylamino or MGS, where "m" represents methylation. cEt-BNA is a restricted ethyl-bridged nucleic acid, PS ASO is a phosphorothioate antisense oligonucleotide, and PO is a phosphodiester bond. Nucleotides at positions 4-6 and 17-19 are cEt BNAs; black indicates a phosphorothioate bond; black / underlined indicates a phosphodiester bond. [Figure 4] Actual fluorescence data from the experiment shown in Figure 3 is shown, demonstrating internalization of the fluorophore into different cell types. [Figure 5] Figure 1 shows siRNA delivery to different cell types with and without conjugation to MGS. [Figure 6] We show that MGS delivery increases the efficacy of antisense oligonucleotides (ASOs) in target cells and reduces their effectiveness in non-target cells. [Figure 7] Immunofluorescence studies show that MGS improves ASO delivery to target cells. DETAILED DESCRIPTION OF THE INVENTION

[0013] The detailed descriptions and examples of specific embodiments contained herein, as well as the drawings and By reference to these contextual descriptions, the disclosed methods and compositions will be more readily understood. It is possible.

[0014] The disclosed methods and compositions do not rely on particular synthetic methods, particular analytical techniques, or It should be understood that the present invention is not limited to any particular reagent or reagents, and may therefore vary. The terminology used herein is for the purpose of describing particular embodiments only. It will be understood that this is not intended to be limiting.

[0015] Preparations thereof that may be used in, in conjunction with, or for the disclosed methods and compositions Disclosed are materials, compositions, and ingredients that can be used in, or are products of, the manufacture of These and other materials are disclosed herein, as are combinations of these materials. When a subset, interaction, group, etc. is disclosed, various individual or Specific reference to each and every combination and variation cannot be expressly disclosed. It is understood that each is specifically contemplated and described herein, even if it is not specifically contemplated. For example, compositions are disclosed and discussed and performed on a number of molecules, including this MGS peptide. When multiple modifications are discussed, each and every combination and permutation of this composition is included. , as well as possible modifications, are specifically contemplated unless specifically indicated to the contrary. That is, a class consisting of molecules A, B, and C and molecules D, E, and F. When AD is disclosed as an example of a combination molecule, each of the classes Even if not individually listed, each combination is contemplated individually and collectively. Therefore, in this example, A, B and C; D, E and F, and the example of combinations of AD From the disclosure, the combination of AE, AF, BD, BE, BF, CD, CE and CF Each combination should be considered specifically contemplated and disclosed. , any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, A, B and C; D, E and F, and AD are examples of combinations. From the disclosure, the subgroups AE, BF and CE are specifically envisaged and disclosed. This concept should be considered in conjunction with the methods of making and using the compositions of the present disclosure. This applies to all aspects of this application, including but not limited to the steps of: If there are various additional steps that can be performed, each of these additional steps may be included in the disclosed method. It is understood that the present invention may be practiced in accordance with any particular embodiment or combination of embodiments, and that such practice may be practiced in accordance with any particular embodiment or combination of embodiments. Each such combination should be considered to be specifically contemplated and disclosed.

[0016] A.Definition The disclosed methods and compositions may vary in the particular methodology, protocols, and reagents described. It is understood that the terms used herein are not limited to specific implementations. It is for illustrative purposes only and is not intended to limit the scope of the present disclosure. It should also be understood that the scope of the present invention is limited only by the appended claims. .

[0017] As used in this specification and the appended claims, the singular forms "a," "an," and " "The" includes plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to "MGS" includes a plurality of such MGSs. Reference to a "nucleic acid sequence" includes one or more nucleic acid sequences known to those of skill in the art and includes those and similarly in other cases.

[0018] As used herein, "treating" refers to administering a composition of the present invention to a subject suffering from a disease or condition. A subject, e.g., a human or other mammal (e.g., an animal model), is treated with the disease or to prevent or delay the worsening of the effects of a medical condition, or to treat the disease or condition is meant to mean administering to partially or completely reverse the effects of In some embodiments, the disease or condition can be cancer. Diseases, disorders and / or or to reduce the risk of developing a condition associated with a disease, disorder, or and / or for subjects who show no signs of a disease, disorder, and / or Treatment may be administered to subjects who show only early signs of disease. In embodiments, treatment involves delivering one or more of the disclosed compositions to a subject.

[0019] As used herein, "prevent" means to reduce susceptibility to the development of a disease, disorder, or condition. Minimizing the likelihood of progression of the disease, disorder, or condition in a subject with increased It is meant to mean that.

[0020] As used herein, the term "subject" refers to the target of administration, e.g., a human. Thus, the subject of the disclosed methods is a vertebrate, e.g., a mammal, a fish, a bird, The term "subject" also includes domestic animals (e.g., reptiles, amphibians, etc.). cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and This also includes laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). In an embodiment, the subject is a mammal. In another embodiment, the subject is a human. Therefore, regardless of sex, it does not refer to adults, children, adolescents, and Newborn subjects, as well as fetuses, are intended to be encompassed.

[0021] As used herein, the term "patient" refers to a subject suffering from a disease or disorder. The term "patient" includes human and animal subjects. Some aspects of the methods of the present disclosure In the present specification, the "patient" has been diagnosed as needing treatment prior to the administering step.

[0022] As used herein, the term "amino acid sequence" refers to a sequence of amino acid residues, abbreviations, letters, or the like. A book means a list of letters, characters, or words. The amino acid abbreviations used in the specification are the conventional single letter codes for amino acids, as follows: They are represented as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid. F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamic acid R, arginine; S, serine; T, threonine; V, valine; W, tryptophan Fan; Y, tyrosine.

[0023] As used herein, "polypeptide" refers to any peptide, oligopeptide, Polypeptide means a gene product, expression product, or protein. Consisting of consecutive amino acids. The term "polypeptide" refers to a naturally occurring or synthetic molecule. Contains.

[0024] Furthermore, as used herein, the term "polypeptide" refers to a polypeptide that is a polypeptide having a peptide bond or modification. means amino acids linked together by peptide bonds (e.g., peptide isosteres), It may contain modified amino acids other than the 0 gene-encoded amino acids. The polypeptides may be synthesized by natural processes, such as post-translational processing, or by other methods well known in the art. The modifications can be made by any of the following chemical modification techniques: It can occur anywhere in a polypeptide, including the chain, and the amino or carboxyl termini. The same type of modification can be present to the same extent at several sites in a given polypeptide. A given polypeptide may be present in many species, or in different degrees. Modifications include acetylation, acylation, ADP-ribosylation, etc. , amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of heme moiety, nucleic acid Covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, Covalent binding of sulfidyllinositol, disulfide bond formation, demethylation, cysteine or pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GP I-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, arylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, Transcriptional RNA-mediated addition of amino acids to proteins, such as sulfation and arginylation These include, but are not limited to, Proteins - Structure and d Molecular Properties 2nd Ed.,TECreig hton,WH Freeman and Company,New York(19 93);Posttranslational Covalent Modificat ion of Proteins, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

[0025] As used herein, the phrase "nucleic acid sequence" refers to a nucleic acid sequence that is a sequence of DNA or RNA or DNA. A-RNA hybrids, single-stranded or double-stranded, sense or antisense and hybridizes to complementary nucleic acids by Watson-Crick base pairing. Naturally occurring or synthetic oligonucleotides or polynucleotides that can The nucleic acid sequences of the present invention also include nucleotide analogs (e.g., BrdU), and non-phosphorylated nucleotides. Phosphodiester internucleoside linkages (e.g., peptide nucleic acids (PNA) or thiodiesters) In particular, nucleic acid sequences may include DNA, RNA, cDNA, gDNA, A, ssDNA, dsDNA or any combination thereof, Not limited to these.

[0026] As used herein, an "effective amount" of a composition is a sufficient amount to produce a desired effect. The exact amount required will vary depending on the species, age, and dosage of the subject. and general condition, the severity of the disease being treated (or the underlying genetic defect), the It will vary between subjects depending on the specific compound used, its method of administration, etc. It is not possible to specify an "effective amount." However, an appropriate "effective" amount is This can be determined by one of ordinary skill in the art using only experimentation.

[0027] As used herein, "selectively binds" refers to a nucleic acid sequence (e.g., cargo) or The MGS recognizes and physically interacts with its target (e.g., a specific cell type). , meaning that it does not specifically recognize or interact with other targets.

[0028] The term "percent homology" is used herein interchangeably with the term "percent identity." used interchangeably and may be used to align sequences with wild-type or control sequences using a sequence alignment program. The term "identity" refers to the level of nucleic acid or amino acid sequence identity when aligned with a sequence other than the one identified. For example, as used herein, 80% identity is 80% as measured by the defined algorithm. This means the same as 80% sequence identity, and therefore a homolog of a given sequence is Having greater than 80% sequence identity over the length of the sequence. Exemplary Levels of Sequence Identity Examples include 8 sequences for a given sequence, e.g., any of the MGS sequences described herein. Examples include 0, 85, 90, 95, 98% or more sequence identity. Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to: The programs include the BLAST suite of programs publicly available on the Internet; For example, BLASTN, BLASTX, and TBLASTX, BLASTP, and T Examples include, but are not limited to, BLASTN. Altschul, et al. See also Altschul, et al., 1990 and Altschul, et al., 1997. Searches are typically performed on nucleic acid sequences in GenBank DNA sequences and other public databases. The BLASTN program is used to evaluate a given nucleic acid sequence against All amino acid sequences in the Bank protein sequence and other public databases To search for a nucleic acid sequence translated into all reading frames, use BLASTX. The programs preferred are BLASTN and BLASTX, both of which are in the OpenGIS 11.0 release. The default parameters for gap penalty and widening gap penalty are 1.0. The method is performed using the BLOSUM-62 matrix (see, e.g., Altschu et al., J. Med. Soc. Chem. Soc ... l,SF,et al.,Nucleic Acids Res.25:3389- 3402, 1997.) To determine "percent identity" between two or more sequences, For this purpose, a preferred alignment of selected sequences is, for example, an open gap of 10.0. Penalty, 0.1 widening gap penalty, and BLOSUM30 similarity matrix Mac Vector version 13.0, which operates with default parameters including This is done using the CLUSTAL-W program in .7.

[0029] Substitutions, deletions, insertions, or any combination thereof may be used to create the final derivative, barrier These changes generally involve the modification of the molecule to a minimum. To keep it to a minimum, changes are made to a few nucleotides. However, more changes are possible. May be acceptable in certain circumstances.

[0030] Generally, the nucleotide identity between individual variant sequences is at least 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % of the parent or reference sequence of the present invention. It can be a sequence with a specified identity to a sequence (e.g., a wild-type sequence), 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 1 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 biological mechanisms, including but not limited to 5%, 96%, 97%, 98%, or 99% For example, a "variant sequence" is a sequence that has a specific function that differs from a parent or reference sequence of the present invention. , 1, 2, or 3, 4 nucleotide base changes that alter the biological function, particularly of the parent sequence. The sequences may be identical and / or have the same or improved activity. In this context, a "variant sequence" is a sequence that has a specified identity to a parent sequence of the present invention. and can have at least 80%, 81%, 82% of the specificity and / or activity of the parent sequence. ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% Variant sequences share unspecified biological functions with the reference sequence (e.g., MGS sequence). Specificity and / or activity of at least 80%, 81%, 82%, 83%, 84%, 85% %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, or 99% can also be shared.

[0031] As used herein, "modulate" means to change by increasing or decreasing. What is meant to be meant.

[0032] "Optional" or "optionally" refers to any event, circumstance, or material described below. may or may not occur, may or may not exist, and the description does not when an event, circumstance, or material occurs or exists, and when such event, circumstance, or material cannot occur This means including cases where the condition is not present or can not exist.

[0033] Ranges are expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, the context may specifically state otherwise. Unless expressly stated, ranges from one particular value and / or to another particular value are also included. Also, what is specifically contemplated and contemplated is disclosed. So, when values ​​are expressed as approximations, it is natural that the context may specifically suggest otherwise. Unless otherwise indicated, specific values ​​may not be used to represent other specifically envisioned implementations that should be considered disclosed. It will further be understood that unless the context specifically indicates otherwise, The endpoints of a range are significant both in relation to the other endpoint, and independently of the other endpoint. Finally, all individual values ​​and subranges of values ​​that fall within any expressly disclosed range are also included. It is also contemplated and disclosed unless specifically contemplated and the context specifically indicates otherwise. It should be understood that the foregoing is indicative of some of these embodiments. Any or all of the provisions of this section apply whether or not they are expressly disclosed in a specific case.

[0034] Unless otherwise defined, all technical and scientific terms used herein refer to the disclosed methods and The terms "composition" and "compositions" have the same meaning as commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described may be used in the practice or application of the methods and compositions of the invention. Although any method, device, and material that may be used for any purpose, particularly useful methods, devices, and materials are described in Publications cited herein and the materials for which they are cited are incorporated by reference. No. 6,317,793, filed Dec. 1, 2002, entitled "Compounds of a Compound Non-Patent That Produced by a Method and Apparatus for Producing and Using the Invention," filed Dec. 1, 2002, and ... Nothing in this publication should be construed as an admission that no rights exist prior to the publication of any No admission is made that any of the references constitute prior art. , assertions by the authors, and applicants challenge the accuracy and pertinence of the documents cited. Many publications are referenced in this specification. Although such references are provided, it is understood that any such documents are common knowledge in the art. It will be expressly understood that this is not an admission that it forms part of general knowledge.

[0035] Throughout the description and claims of this specification, the word "comprises" may be used. The word "comprising" and variations of that word, such as "comprising" and "including "(comprises)" means "including but not limited to," e.g. For example, it is not intended to exclude other additions, components, integers or steps. A method described as including the above steps or operations may include what is indicated for each step. Each step is specifically contemplated (unless the step includes a limiting term such as "consisting of"). A process may, for example, exclude other additions, components, integers, or steps not shown in the process. means that it is not intended to

[0036] B. Composition Disclosed are compositions comprising cargo conjugated to one or more MGS peptides. In some embodiments, the cargo is a nucleic acid. Thus, it can be conjugated to one or more MGS peptides. Disclosed are compositions comprising the conjugated nucleic acid sequences. In some embodiments, the disclosed compositions comprise , can be used for transport across lipid membranes, and therefore can be used as a transmembrane composition or conjugate. It can point to

[0037] The components used to make the disclosed compositions are These and other materials are disclosed herein as well as the compositions themselves used therein. and combinations, subsets, interactions, groups, etc. of these materials are disclosed. When these compounds are used in various combinations and Specific references to the various arrangements and permutations may not be explicitly disclosed, but each It is understood that this is specifically contemplated and described herein.

[0038] 1.MGS peptide MGS peptides or targeting peptides are disclosed herein. These peptides include: The MGS peptides that can be used or modified in the disclosed compositions are capable of selectively binding to cells. Examples of petids include McGuire et al., Sci Rep. 2014 Ma r 27;4:4480 MG can also be used in the disclosed compositions and methods. Examples of S peptides include, but are not limited to, the MGS sequences shown in Table 1. .

[0039] [Table 1-1] [Table 1-2]

[0040] In some embodiments, the one or more MGS peptides are CSAGT (SEQ ID NO: 9); LQWRRDDNVHNFGVWARYRL (SEQ ID NO: 20 );RGDLATLRQLAQEDGVVGVR (SEQ ID NO: 1);ATEPRKQYAT PRVFWTDAPG (SEQ ID NO: 13); FHAVPQSFYTAP (SEQ ID NO: 17); EHPWFNMWSWATQVQE (SEQ ID NO: 30), or a combination thereof Includes:

[0041] In some embodiments, the one or more MGS peptides are CSAGT (SEQ ID NO: 9); LQWRRDDNVHNFGVWARYRL (SEQ ID NO: 20 );RGDLATLRQLAQEDGVVGVR (SEQ ID NO: 1);ATEPRKQYAT PRVFWTDAPG (SEQ ID NO: 13); FHAVPQSFYTAP (SEQ ID NO: 17); or EHPWFNMWSWATQVQE (SEQ ID NO: 30) and at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity In some embodiments, one or more MGS peptides have a sequence identity of Any of the disclosed MGS peptides and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 114 having a sequence identity of 4%, 95%, 96%, 97%, 98%, or 99% identity.

[0042] In some embodiments, the MGS peptide can be optimized. For example, SEQ ID NO: 3 , 4, 5, 6, 7, 8, 11, 12, 15, 16, 18, 19, and 22 are optimized. Modifications were made to individual parent peptide sequences identified by the FOX-3 platform technology. These modifications were used to obtain optimized peptides. Identify essential amino acids in the parent sequence required for binding and internalization. These modifications are Combined alanine scanning and truncation of the amino- and c-terminal regions of the parent peptide PEG11 provides protection for the C-terminus of the MGS peptide, providing a spacer between the peptide and the cargo molecule linked via the cysteine, Increases the solubility of MGS-peptides, making them less susceptible to degradation by peptidases in the blood. (Leu) protection, etc., by acetylation (CH3CO-) and / or d-amino acids, Modification at the amino terminus. Optimized peptides of uniform length applicable to all MGS peptides. No standard was used, and all changes were tested to determine their effect on peptide uptake and stability. In peptide 73, we used YC to synthesize peptides and The concentration can be monitored by absorbance at 280 nm. Tyrosine (Y) is added. Without it, the peptide becomes significantly more difficult to monitor. CH3CO-YAAWPASGAWT-PEG 11 -C-NH2 (SEQ ID NO: 12), CH3 CO-LQWRRNFGVWARYRL-PEG 11 -C-NH2 (SEQ ID NO: 22), CH 3CO-RGDLATLRQL-PEG 11 -YC-NH2 (SEQ ID NO: 4), CH3CO- RGDLATLRQL-PEG 11 -Y-NH2 (SEQ ID NO: 5), CH3CO-d(Leu) -RGDLATLRQL-PEG 11 -YC-NH2 (SEQ ID NO: 6), CH3CO-d(Le u)-RGDLATLRQL-PEG 11 -Y-NH2 (SEQ ID NO: 7), CH3CO-KQY ATPRVFWT-PEG 11 -C-NH2 (SEQ ID NO: 16), or CH3CO-FHAV PQSFYT-PEG 11 -C-NH2 (SEQ ID NO: 19) Thus, in some embodiments, one or more MGS peptides of the disclosed compositions , CH3CO-YAAWPASGAWT-PEG 11 -C-NH2 (SEQ ID NO: 12), CH3 CO-LQWRRNFGVWARYRL-PEG 11 -C-NH2 (SEQ ID NO: 22), CH 3CO-RGDLATLRQL-PEG 11 -YC-NH2 (SEQ ID NO: 4), CH3CO- RGDLATLRQL-PEG 11 -Y-NH2 (SEQ ID NO: 5), CH3CO-d(Leu) -RGDLATLRQL-PEG 11 -YC-NH2 (SEQ ID NO: 6), CH3CO-d(Le u)-RGDLATLRQL-PEG 11 -Y-NH2 (SEQ ID NO: 7), CH3CO-KQY ATPRVFWT-PEG 11 -C-NH2 (SEQ ID NO: 16), or CH3CO-FHAV PQSFYT-PEG 11 -C-NH2 (SEQ ID NO: 19), or a combination thereof It is possible.

[0043] In some embodiments, one or more MGS peptides are CH3CO-YAAWPASGA WT-PEG 11 -C-NH2 (SEQ ID NO: 12), CH3CO-LQWRRNFGVWAR YRL-PEG 11 -C-NH2 (SEQ ID NO: 22), CH3CO-RGDLATLRQL- PEG 11 -YC-NH2 (SEQ ID NO: 4), CH3CO-RGDLATLRQL-PEG11 -Y-NH2 (SEQ ID NO: 5), CH3CO-d(Leu)-RGDLATLRQL-PEG 11 -YC-NH2 (SEQ ID NO: 6), CH3CO-d(Leu)-RGDLATLRQL-P EG 11 -Y-NH2 (SEQ ID NO: 7), CH3CO-KQYATPRVFWT-PEG 11 -C -NH2 (SEQ ID NO: 16), or CH3CO-FHAVPQSFYT-PEG 11 -CN H2 (SEQ ID NO: 19) and at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% identity.

[0044] In some embodiments, one or more MGS peptides are modified by acetylation at the N-terminus. Thus, in some embodiments, one or more MGS peptides can be acetylated. In some embodiments, one or more MGS peptides can be synthesized by the nucleic acid sequence In some embodiments, the chemical conjugate can be polyethylene glycol (PEG). Thus, in some embodiments In some embodiments, one or more of the MGS peptides can be PEGylated. The number of PEG units can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or any combination thereof. In some embodiments, the number of PEG units can be one or more MG S peptide from the nucleic acid sequence, and The length of the bond can be long enough to prevent any steric interference. 1. A composition comprising a chemical conjugate, wherein the chemical conjugate is PEG, and the PEG is 11 Disclosed herein is a composition comprising one or more PEG units. The MGS peptide comprises SEQ ID NO: 9 or 10, which has an N-terminal amino acid sequence similar to that of SEQ ID NO: 9 or 10. It can be acetylated, chemically conjugated to PEG, and can be used in nucleic acid sequences. The string can be covalently attached to the PEG.

[0045] In one embodiment, one or more of the MGS peptides disclosed herein can be truncated. In some embodiments, active portions of MGS peptides can be used in the disclosed compositions. In some embodiments, the active moiety can be prepared by techniques well known in the art, e.g., arabinopeptides. The activity of the MGS peptide can be measured using Ninscanning or cleavage studies. The moiety retains the ability to bind to a specific cell and target a specific location within that cell. For example, SEQ ID NO: 9 is truncated by at least 9 amino acids at the C-terminus. Thus, the 11-mer of SEQ ID NO: 9, described herein as SEQ ID NO: 10, can be can be used as the MGS peptide. In some embodiments, SEQ ID NO: 13 is , truncated by about 3, 4, or 5 amino acids at the N-terminus and / or C-terminus. can.

[0046] Any of the MGS peptides listed in Table 1, or a combination thereof, is disclosed. Thus, in some embodiments, one or more MGS peptides are selected from the group consisting of SEQ ID NOS: 1-55. It may include any one of these, or a combination thereof.

[0047] In some embodiments, the composition can include one or more MGS peptides. For example, in some embodiments, the composition comprises 1, 2, 3, 4, or 5 MGS peptides. In some embodiments, one or more MGS peptides may be a tetrameric scaffold. In some embodiments, the MGS peptide can form a monomelic protein. In some embodiments, multiple MGS peptides, e.g., For example, but not limited to, dimers or trimers may be used together. In the case of 2 or more MGS peptides, 2 or more MGS peptides can be used together. In some embodiments, two or more MGS peptides can have the same sequence. Two or more MGS peptides may be used together and may have different sequences. In some embodiments, a plurality of MGS peptides, e.g., a dimer, comprises two or more identical MGS peptides. In some embodiments, the plurality of MGS peptides, e.g., the dimer, comprises at least one of the following sequences: also contains two different MGS sequences.

[0048] In some embodiments, one or more MGS peptides localize one or more intracellular targets. For example, the intracellular target may be the lysosome, the Golgi apparatus, the endoplasmic reticulum, the cytoplasm, or the nucleus. Any intracellular compartment can be targeted, including, but not limited to: .

[0049] The FOX-Three platform allows rapid screening against target cells Capable, 10 9 ~10 12 Established MGS members based on candidate peptides Phage display libraries and methods for identifying specifically targeted MGSs Based on the intracellular system (Figure 2), peptides are synthesized by biological processes and artificial chemical processes. It is a well-understood class of biomolecules that can be rapidly synthesized by processes. The power of the FOX-Three platform is demonstrated in its speed and flexibility. It is applicable to any cell type, regardless of knowledge of the molecular characteristics of that cell. The selected peptide-based MGS is then used to generate the lead MGS in 2-4 weeks. Table 2 shows the results of the FOX-Three technology identified in the This is MGS.

[0050] [Table 2-1] [Table 2-2]

[0051] In some embodiments, one or more MGS peptides are used to identify proteins that are generally linked to nucleic acids. It can be conjugated to a nucleic acid sequence either by conjugation or by binding. In some embodiments, the nucleic acid sequence is linked to one or more MGS peptides via a linker. For example, in some cases, the linker can be a peptide linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the MGS peptide can be a covalent bond, a bifunctional linker, or a using any known method, including but not limited to conjugation chemistry. and can be conjugated to a nucleic acid sequence.

[0052] In some embodiments, the cargo molecule is conjugated to one or more MGS sequences. In some embodiments, the cargo molecule is a nucleic acid sequence, a protein, an antibody, a peptide, The nanoparticles can be, but are not limited to, nanoparticles, dyes, or small molecules. As described herein, the cargo is a nucleic acid sequence.

[0053] 2. Nucleic acid sequence In some embodiments, the nucleic acid sequences of the disclosed compositions are DNA, RNA, or DNA / RNA. In some embodiments, the nucleic acid sequence is an antisense nucleic acid. In some embodiments, the nucleic acid sequence is an siRNA or miRNA. A. In some embodiments, the nucleic acid sequence can be any nucleic acid therapeutic agent.

[0054] In some embodiments, the nucleic acid sequence targets an intracellular target. For example, the intracellular target is a can be, but are not limited to, the endoplasmic reticulum, the golgi apparatus, the endoplasmic reticulum, the cytoplasm, or the nucleus No. Any intracellular compartment can be targeted.

[0055] In some embodiments, the nucleic acid sequence is 10-30 nucleotides in length. In some embodiments, the nucleic acid sequence is 10 to 50 nucleotides in length. In some embodiments, the nucleic acid sequence is between 8 and 50 nucleotides in length. In some embodiments, the nucleic acid sequence is 5 to 50 nucleotides in length. In embodiments, the nucleic acid sequence is 5 to 250 nucleotides in length. In some embodiments, the nucleic acid sequence is 5 to 500 nucleotides in length. In some embodiments, the size of the nucleic acid is not limited.

[0056] In some embodiments of the disclosed compositions, the nucleic acid sequence can further comprise a label. For example, the compositions disclosed herein can include a detectable label. Alternatively, the label can be a fluorescent dye. a tag designed for detection (e.g., purification or localization) of the polypeptide or sequence being studied Examples of tag sequences include, but are not limited to, green fluorescent protein sequences. Photoprotein, glutathione S-transferase, polyhistidine, c-myc, heme maglutinin, or Flag™ tag, which can be fused to the encoding nucleic acid. Such detectable labels can include fluorescent agents, enzyme labels, or radioisotopes. These include, but are not limited to, the body.

[0057] The compounds of the present invention that are present in the compositions described herein and administered to mammals (e.g., humans) The therapeutically effective amount of nucleic acid used in the disclosed methods will depend on age, weight, and other factors (as discussed above). This can be determined by a person skilled in the art taking into account individual differences in other general conditions. These compositions can be stable in serum and bloodstream and, in some cases, more specific, making them The dose of a composition containing any individual component is the dose of any of the individual components when uncombined. The effective amount may be less than (or more than) the above.

[0058] In some embodiments, the nucleic acid sequence is T m CAG m CATT m CTAATAG m CAG m C(distribution SEQ ID NO: 56 is the sequence for MALAT 1. .

[0059] 3. Pharmaceutical Compositions In some embodiments, the disclosed compositions can be pharmaceutical compositions. In some embodiments, compositions comprising nucleic acid sequences conjugated to one or more MGS peptides are provided. and a pharmaceutically acceptable carrier. The formulation may be formulated to minimize and counter any degradation of the active ingredient, as is well known to those skilled in the art. "means a material or carrier selected to minimize any adverse side effects in the subject" Examples of carriers include dimyristoyl phosphatidylcholine (DMPC), phosphate buffered saline, and Physiological saline, or multivesicular liposomes. For example, PG:PC:cholesterol Le:peptide or PC:peptide may be used as a carrier in the present invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19 th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, An appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Other examples of carriers include saline, Ringer's solution, and dextrose solution. The pH of the solution may be, but is not limited to, about 5 to about 8, or about 7 to about 7.5. Further carriers can be solid hydrophobic polymers containing the composition. Sustained-release preparations include semipermeable matrices, which matrices may be shaped Articles such as films, stents (which can be implanted into vessels during angioplasty procedures), In the form of liposomes or microparticles. For example, the route of administration and the composition being administered It will be apparent to those of ordinary skill in the art that certain carriers may be more preferable depending on the concentration of the compound. Most typically, these are sterile water, saline, and buffered saline at physiological pH. Standard carriers for administration of drugs to humans would include solutions such as buffer solutions.

[0060] Pharmaceutical compositions are intended to achieve the intended activity of the polypeptides, peptides, or conjugates of the present invention. Carriers, thickeners, diluents, buffers, preservatives, etc. may also be included as long as their functionality is not impaired. The pharmaceutical composition can contain antibacterial agents, anti-inflammatory agents, anesthetics, etc. (in the composition of the present invention). In addition, one or more active ingredients may also be included.

[0061] The pharmaceutical compositions disclosed herein can be formulated for oral or parenteral administration. Pharmaceutical compositions prepared for parenteral administration include intravenous (or intraarterial), intramuscular, subcutaneous, and intravenous. , intraperitoneally, transmucosally (e.g., intranasally, intravaginally, or rectally), or transdermally (e.g., topically) The fusion protein may be delivered by aerosol inhalation or other suitable means. Thus, aqueous carriers such as water, buffered water, saline, Acceptable carriers include, but are not limited to, buffered saline (e.g., PBS), and the like. A composition is prepared for parenteral administration containing the fusion protein dissolved or suspended in a carrier. One or more of the excipients included may be, for example, pH adjusting agents and buffering agents, Substances that aid in maintaining approximate physiological conditions, such as osmotic pressure regulators, wetting agents, and surfactants, When the composition contains a solid component (which may be for oral administration), the excipients may be One or more of the following may function as binders or fillers (e.g., for formulations such as tablets, capsules, etc.) When the composition is formulated for application to the skin or mucosal surfaces, One or more of the excipients may be a solvent or emulsifier for formulations such as creams, ointments, etc. .

[0062] The pharmaceutical compositions may be sterile, or may be sterilized by conventional sterilization techniques, or They may be sterile filtered. The aqueous solutions may be packaged for ready use or lyophilized. The lyophilized preparations encompassed by the present disclosure can be dissolved in a sterile aqueous carrier prior to administration. The pH of the pharmaceutical composition is usually 3 to 11 (e.g., about 5 to 9). ) or 6 to 8 (for example, about 7 to 8). The resulting composition in solid form is then prepared as a tablet or A plurality of capsules, each containing a fixed amount of the above drug(s), The composition in solid form may be packaged in a single dose unit. or in squeezable tubes designed for ointments, for flexible dosing. It can also be packaged in a container.

[0063] The pharmaceutical compositions are formulated to contain a therapeutically effective amount of the compositions disclosed herein. In some embodiments, therapeutic administration includes prophylactic applications. Based on clinical and other prognostic factors, the physician consulting the patient should determine whether the patient has one or more autoimmune Clinically measured increased predisposition or susceptibility to a disease (sometimes called susceptibility) or if the patient has a clinically measured predisposition to cancer. or increased susceptibility (in some cases, a large increase in susceptibility), You can select:

[0064] The pharmaceutical compositions described herein may delay, reduce, or preferably prevent the onset of clinical disease. and administering to a subject (e.g., a human subject or human patient) in an amount sufficient to prevent or Thus, in some embodiments, the subject is a human subject. The compositions may be administered to subjects already suffering from or diagnosed with an autoimmune disease (e.g., for example, a human subject) to at least partially ameliorate a sign or symptom of a disease state or an amount sufficient to inhibit (and preferably arrest) the progression of the condition, its complications, and consequences An amount adequate to accomplish this is defined as a "therapeutically effective amount." A therapeutically effective amount of a pharmaceutical composition may be an amount that achieves a cure, although that outcome may be achieved As shown, the therapeutic effective dose is the dose that reduces the incidence or severity of cancer. Slowing, hindering, or preventing the progression of, or an autoimmune disease or The present invention relates to a method for treating a rheumatoid arthritis, including administering to a patient a rheumatoid arthritis, an allergic reaction, or an allergic reaction to a rheumatoid arthritis. Treated individuals may experience accelerated recovery.

[0065] The total therapeutically effective amount of the conjugate in the pharmaceutical compositions disclosed herein is determined as follows: administered to mammals as a single dose either by infusion or over a relatively short period of time. or multiple doses over a longer period (e.g., 4-6, 8-12, 1 Every 4-16, or 18-24 hours, or every 2-4 days, or every 1-2 weeks, or It can be administered using a split treatment protocol in which the dose is administered once a month, or Continuous intravenous infusion sufficient to maintain a therapeutically effective concentration in the blood is also within the scope of this disclosure. is.

[0066] C. Method Methods of using the disclosed compositions are disclosed. It can be used in the manner disclosed herein.

[0067] administering to the cells a composition comprising a nucleic acid sequence conjugated to one or more MGS peptides; 1. A method for reducing gene expression of a gene of interest, comprising: RNA transcribed from the target gene, the target gene, or the target In some embodiments, the nucleic acid The sequences include, but are not limited to, antisense oligonucleotides, aptamers, and It can be any therapeutic nucleic acid, such as a small interfering RNA. The reduction in gene expression is determined by measuring protein levels. A decrease in expression leads to a decrease in protein levels.

[0068] administering to the cells a composition comprising a nucleic acid sequence conjugated to one or more MGS peptides; 1. A method for increasing gene expression of a gene of interest, comprising: The method of binding to the target gene or a sequence upstream of the target gene. Disclose the law.

[0069] Thus, cells containing a nucleic acid sequence conjugated to one or more MGS peptides can be used. A method for modifying gene expression of a gene of interest, comprising administering a composition comprising the above-described The nucleic acid sequence may be an RNA transcribed from the gene of interest, the gene of interest, or The method further comprises: binding to a sequence upstream of the gene of interest; In such cases, modifying gene expression of a gene of interest results in increased gene expression of the gene of interest. or a decrease.

[0070] In some embodiments, the sequence upstream of the gene of interest is a sequence encoding the gene of interest. In some embodiments, the gene of interest can be a region of DNA responsible for regulating expression. Binding to the upstream sequence of the molecule increases or decreases gene expression of the target gene. This can be done.

[0071] administering to the cells a composition comprising a nucleic acid sequence conjugated to one or more MGS peptides; a method for targeting a gene of interest, comprising: targeting an intracellular target, wherein the nucleic acid sequence is transcribed from the gene of interest within the intracellular target; In some embodiments, the intracellular target binds to an RNA that is expressed in a lysosome. The membrane may be, but is not limited to, the membrane in the cytoplasm, the golgi apparatus, the endoplasmic reticulum, the cytoplasm, or the nucleus. I can't.

[0072] In some aspects of the disclosed methods, the gene of interest is a gene whose expression or overexpression is It can be any gene that is harmful to the cell or is involved in a disease process. For example, the gene of interest may be expressed in a manner that targets a disclosed intracellular target (e.g., lysosome, Golgi, over- or under-expression in one of the major cellular components (the endoplasmic reticulum, cytoplasm, or nucleus) can cause disease. In some embodiments, the gene of interest can be a gene encoding KRAS or The gene encoding the MYC gene can be, but is not limited to, MYC or MYC.

[0073] A method of treating a subject in need thereof, comprising administering to the subject in need thereof an effective amount of: administering a nucleic acid sequence conjugated to one or more MGS peptides, The method is disclosed, wherein the GS peptide targets an intracellular target involved in the disease process. In some embodiments, the subject in need thereof is a patient suffering from an infectious disease, cancer, diabetes, nervous system or neurological disorders. Do you have a degenerative, inherited, lysosomal, or mitochondrial disease? have been exposed to bioterrorism agents.

[0074] In some embodiments, the disclosed methods involve the use of one or more of the MGS peptides listed in Table 1. You can be there.

[0075] In some embodiments, the intracellular target is a lysosome, a Golgi apparatus, an endoplasmic reticulum, a cytoplasm, or can be, but is not limited to, a nucleus.

[0076] In some embodiments, the nucleic acid sequence is transcribed from a gene of interest within the intracellular target. In some embodiments, the target gene binds to the RNA transcribed from the gene of interest. The binding of the nucleic acid sequence results in a decrease in the expression of the gene of interest. In embodiments, the nucleic acid sequence binds to a gene of interest within the intracellular target. In embodiments, the nucleic acid sequence binds to a sequence upstream of a gene of interest within an intracellular target. In some embodiments, the sequence upstream of the gene of interest is a sequence that regulates gene expression of the gene of interest. In some embodiments, the gene of interest can be a region of DNA responsible for regulating Binding to the upstream sequence of the target gene increases or decreases gene expression. can be done.

[0077] In some embodiments, the gene of interest is a gene whose under- or over-expression contributes to a disease process. For example, the gene of interest may be any gene that is involved in the expression of a disclosed intracellular target (e.g., excess or in one of the following tissues: lysosomes, Golgi apparatus, endoplasmic reticulum, cytoplasm, or nucleus In some embodiments, the gene may be an underexpressed gene that causes a disease. The gene can be, but is not limited to, KRAS or MYC.

[0078] In some embodiments, the disclosed methods of treatment include administering one or more additional therapeutic agents. In some embodiments, the disclosed compositions can be used alone or in combination with other In some embodiments, the disclosed The compositions may be administered alone or in combination with one or more additional therapeutic agents (e.g., biologically active agents). They can be formulated together into a composition suitable for administration to a subject. Methods for treating a subject having or at risk of developing cancer, as disclosed herein. The compositions can be administered, for example, in a therapeutically effective amount of radiation therapy, immunotherapy, or chemotherapy, or The combination therapy can be administered as a co-preparation or When administered separately, the combination therapy may be administered simultaneously or sequentially. The formulations can be prepared using methods routine in the art. It can be made with.

[0079] D. Vector Disclosed are vectors comprising nucleic acid sequences encoding one or more of the disclosed compositions. In one embodiment, the vector comprises a nucleic acid sequence capable of encoding one or more of the disclosed MGS peptides. Includes

[0080] E. Kit The materials described above, as well as other materials, can be combined into kits useful for carrying out the disclosed methods, may be combined in any suitable combination as a kit to aid in the performance of the disclosed methods. The kit components in a given kit may be packaged in the manner disclosed. It is useful when it is designed and adapted for use with the method, for example: Kits containing one or more of the disclosed compositions are disclosed.

[0081] In some embodiments, the disclosed kits comprise one or more of the disclosed MGS peptides, and / or Alternatively, it may comprise one or more of the disclosed nucleic acid sequences. [Example]

[0082] The FOX-Three platform is already established for targeting cells and subcellular components. The numerous MGSs identified have been used in a variety of different cell targeting systems. Currently in development stages (Table 1). Methods for conjugating various payloads to peptide MGS The MGS selected have been established without destroying their targeting ability. Delivery of drugs, nanoparticles, DNA, and proteins to target cells in culture and animal models The FOX-Three imaging process has been refined in recent years, and This includes targeting all intracellular locations, including lysosomes, autophagosomes, and other intracellular locations within cancer cells. In addition to accumulating in the nucleus and Golgi, MGSs that target the plasma membrane have been isolated. The therapeutic effect of S has been shown to depend on delivery to the correct intracellular location.

[0083] With sufficient cell types, disease states, subcellular organelles, and deliverable payloads, FOX The potential of the -Three platform is vast. Creating an optimized MGS "toolbox" capable of delivering cytosolic and intracellular components To do this, FOX Expanding the number of targetable subcellular organelles using the -Three platform A series of experiments with quantitative milestones can be performed.

[0084] In some embodiments, non-small lung cancer cell lines are targeted to lysosomes, Golgi, and mitochondria. The synthesis, characterization, and optimization of three MGSs that accumulate in endothelia can be studied. .

[0085] Furthermore, the intracellular locations have been expanded to include lysosomes, Golgi, mitochondria, endoplasmic reticulum, and cells. The material and the core can be included.

[0086] FOX-Three is rapidly developing intracellular targeting of human therapeutic antibodies at unprecedented speed. Discover and provide intelligence to create a highly secure and rapid response to emerging threats .

[0087] The developed FOX-Three MGS toolbox will be used to deliver a variety of other payloads. While delivery has been the primary focus, MGS is able to deliver many different payloads. It is important to note that the drug may deliver steroids and have other clinical uses (Table 1 ). MGS is a powerful tool for detecting small molecule drugs, contrast agents, nanoparticles, DNA, radionuclides, and other proteins. MGS can be used for early detection of disease and as a companion diagnostic. Early disease detection is often the primary determinant of clinical outcome. Companion diagnostics can follow the response to treatment and can be used to assess the rapid progression of treatment. This allows for rapid change, saving time and money when needed. Drugs can be used for targeted therapeutics (small molecules, nucleic acids). MGS therapeutics can be used for various Personalized therapeutics for a variety of disease states, in vitro and in vivo diagnostics, and cancer Targeted therapeutics (small molecules, nuclear) for intracellular nanoparticle delivery and innovative immunotherapy can be used for a variety of polymers (e.g., acids, synthetic macromolecules such as polymers, and proteins).

[0088] [Table 3]

[0089] The following is the structure of the multimerization core, where R is the covalent bond to the cysteine ​​via the sulfhydryl group. R' represents a conjugated MGS. binding sites for proteins, other peptides, drugs, nanoparticles, imaging agents, nucleic acids, or dyes Conjugation can be achieved via maleimide chemistry, click chemistry, or amide chemistry. and via hydrazones. [ka]

[0090] Dimers can also be synthesized using linear peptide chemistry (without maleimide groups). The following can be prepared using linear peptide chemistry (without maleimide groups): An example of the structure of the prepared dimer is shown below: , proteins, other peptides, drugs, nanoparticles, imaging agents, nucleic acids, or dyes) Represents a place. ] [ka]

[0091] 1. Example 1: Effective dose, stoichiometry, and uptake rate for MGS2. Table 4 below shows the EC50 values ​​for MGS on various cell lines. The internalized molecules ranged from about 50,000 to about 125,000, with half-lives ranging from 11 to 37 minutes. It has a reduction life.

[0092] [Table 4]

[0093] Figure 3 shows the results of four different cell cultures at two different temperatures and with a control and without MGS. Thus, Figure 4 shows the internalization of ATP into cells at two different temperatures. This indicates the presence of a segmented fluorophore.

[0094] 2. Example 2: MGS and Nucleic Acids as Cargo The idea of ​​MGS was applied to nucleic acids, which are known to regulate gene expression. are good candidates for therapeutics, but their poor pharmacological properties (e.g., large size) Due to their high charge, rapid clearance from the body, nucleic acid therapeutics are Progress has been slow. MGS is applicable to nucleic acids and supports the internalization of therapeutic nucleic acids. If we can help, this could open the door to using nucleic acids in new therapeutic approaches. The door will open.

[0095] To this end, a novel MGS was applied to facilitate cell-specific delivery of siRNA. As previously mentioned, these MGSs possess specific binding domains that bind to specific epithelial-derived cancer cell types. Upon binding, they are rapidly internalized and trafficked to specific intracellular locations. Chemically optimized MGS has an affinity of 1-2 nM for its cellular target. activity, serum stability for over 48 hours, and increased activity of targeted cancer cells over normal control cells The target cells react with these peptide ligands with approximately 50 to 1000 times more specificity. The drug is quickly internalized and administered within approximately 10 to 30 minutes. 1 / 2 and reach a cell concentration of 1.5 μM or less. Figure 5 shows the effect of siRNA on MGS-free cells and either the 5' or 3' end of the siRNA. Comparison of siRNA delivery to cells containing biotinylated MGS in the following experiments is shown. was first performed using H1993 cells. The bar group represents 5' biotinylated siRNA + SA-647 and 3' biotinylated siRNA without MGS. The bars in the center and on the far left are SA- 647, 5' biotinylated siRNA + SA-647, and the first MGS (SRI_ MGS1_V4, right) and the second MGS (SRI_MGS2_V4, center). The figure shows the 3' biotinylated siRNA + SA-647 conjugated to the 3' biotinylated siRNA. By contrast, siRNA was not internalized in H1993 cells. By conjugating iRNA to SRI_MGS1_V4, the data show that three si For all forms of RNA, approximately 25,000 molecules were internalized per cell. The conjugation of three different forms of siRNA to SRI_MGS2_V4 was A better understanding of the approximately 150,000 to 210,000 molecules internalized per cell Similar results were confirmed in experiments using H1299 cells, and , internalization is close to zero for the MGS-free siRNA form, and SRI_MGS2_ For the V4-conjugated siRNA form, the approximate internalization was approximately 80.0 00 to approximately 95,000.

[0096] In summary, this technology demonstrates the ability to rapidly generate MGS-targeting agents that target pathogen-infected cells. and improves treatment against existing and emerging pathogens in weeks versus years. technology is too slow to develop targeted treatments against evolving viruses. do not have.

[0097] This technology establishes an entirely new arsenal for protecting soldiers, using previously cell-impermeable A new class of therapeutic agents for the treatment of numerous diseases by delivering compounds previously undetectable in vivo It will be opened.

[0098] The technology is otherwise reactive or diffuse / re-diffuse through the community. This provides the ability to detect and neutralize potential intracellular viral infections that may be virulent.

[0099] This technology will rapidly develop MGS that can be used in diagnostic and sensor technologies for biological indicators. It can be developed.

[0100] Various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. It will be apparent to one skilled in the art that other aspects of the invention are possible. These and other modifications will become apparent to those skilled in the art upon consideration of the practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only and not to limit the true scope and spirit of the invention. The scope of the invention is set forth in the following claims.

[0101] Those skilled in the art will recognize many equivalents to the specific embodiments of the methods and compositions described herein. Able to recognize or identify objects without more than conventional experimentation. Such equivalents are intended to be encompassed by the following claims.

Claims

1. Nucleic acid sequences conjugated to one or more molecular guidance system (MGS) peptides A composition comprising:

2. The composition of claim 1 , wherein the nucleic acid sequence is an antisense oligonucleotide.

3. The composition of claim 1 or 2, wherein the nucleic acid sequence targets an intracellular target.

4. The one or more MGS peptides have the sequence: YAAWPASGAWTGTAPCSAGT (SEQ ID NO: 9); LQWRRDDNVHNFGVWARYRL (SEQ ID NO: 20); RGDLATLRQLAQEDGVVGVR (SEQ ID NO: 1); ATEPRKQYATPRVFWTDAPG (SEQ ID NO: 13); FHAVPQSFYTAP (SEQ ID NO: 17); EHPWFNMWSWATQVQE (SEQ ID NO: 30); or a combination thereof The composition according to any one of claims 1 to 3, comprising:

5. The one or more MGS peptides may be selected from the group consisting of: YAAWPASGAWTGTAPCSAGT (sequence No. 9), LQWRRDDNVHNFGVWARYRL (SEQ ID NO: 20), RGDLAT LRQLAQEDGVVGVR (SEQ ID NO: 1), ATEPRKQYATPRVFWTDA PG (SEQ ID NO: 13), FHAVPQSFYTAP (SEQ ID NO: 17), EHPWFNMW SWATQVQE (SEQ ID NO: 30), or a combination thereof with at least 95% to 9 The composition of any one of claims 1 to 3, having 9% sequence identity.

6. 6. The method according to claim 1, wherein the one or more MGS peptides are acetylated. The composition according to item .

7. 7. Any one of claims 1 to 6, wherein the one or more MGS peptides are PEGylated. The composition described in

8. The one or more MGS peptides have the sequence: CH 3 CO-YAAWPASGAWT-PEG 11 -C-NH 2 (SEQ ID NO: 12); CH 3 CO-LQWRRNFGVWARYRL-PEG 11 -C-NH 2 (SEQ ID NO: 22 ）； CH 3 CO-RGDLATLRQL-PEG 11 -YC-NH 2 (SEQ ID NO: 4); CH 3 CO-d(Leu)-RGDLATLRQL-PEG 11 -YC-NH 2 (Array number No. 6); CH 3 CO-d(Leu)-RGDLATLRQL-PEG 11 -Y-NH 2 (SEQ ID NO: ７）； CH 3 CO-KQYATPRVFWT-PEG 11 -C-NH 2 (SEQ ID NO: 16); CH 3 CO-FHAVPQSFYT-PEG 11 -C-NH 2 (SEQ ID NO: 19); or A combination of these The composition according to any one of claims 6 to 7, comprising:

9. The one or more MGS peptides 3 CO-YAAWPASGAWT-PEG 11 - C-NH 2 (SEQ ID NO: 12); CH 3 CO-LQWRRNFGVWARYRL-PEG 11 - C-NH 2 (SEQ ID NO: 22); CH 3 CO-RGDLATLRQL-PEG 11 -YC-NH 2 (SEQ ID NO: 4); CH 3 CO-d(Leu)-RGDLATLRQL-PEG 11 -YC- NH 2 (SEQ ID NO: 6); CH 3 CO-d(Leu)-RGDLATLRQL-PEG 11 -Y -NH 2 (SEQ ID NO: 7); CH 3 CO-KQYATPRVFWT-PEG 11 -C-NH 2 ( SEQ ID NO: 16); CH 3 CO-FHAVPQSFYT-PEG 11 -C-NH 2 (SEQ ID NO: 1 9); or a combination thereof, The composition according to any one of claims 6 to 7.

10. The nucleic acid sequence is conjugated to one or more MGS peptides via a linker. The composition according to any one of claims 1 to 9.

11. The composition of any one of claims 1 to 10, wherein the nucleic acid sequence comprises a label.

12. The composition of claim 11 , wherein the label is a fluorescent dye.

13. 13. The method according to claim 1, wherein the nucleic acid sequence is 10 to 30 nucleotides in length. The composition described.

14. 14. The nucleic acid sequence of claim 1, wherein the nucleic acid sequence is DNA or RNA. composition.

15. administering to a cell a nucleic acid sequence conjugated to one or more MGS peptides. a method for reducing gene expression of a gene of interest, wherein the nucleic acid sequence is A method in which the protein binds to RNA transcribed from a gene that is a target of the protein.

16. administering to a cell a nucleic acid sequence conjugated to one or more MGS peptides. A method for targeting a gene of interest within a cellular target, comprising: , targeting the intracellular target, wherein the nucleic acid sequence is capable of targeting the gene of interest within the intracellular target. A method of binding to RNA transcribed from

17. the intracellular target is a lysosome, a Golgi apparatus, an endoplasmic reticulum, a cytoplasm, or a nucleus. Item 17. The method according to item 16.

18. The gene of interest is a gene whose expression or overexpression is harmful to the cell.

18. The method of claim 16 or 17, wherein the

19. A method of treating a subject in need of treatment, comprising administering to the subject in need of treatment one or more administering an effective amount of a nucleic acid sequence conjugated to the above MGS peptide, A method in which the MGS peptide targets an intracellular target involved in a disease process.

20. the intracellular target is a lysosome, a Golgi apparatus, an endoplasmic reticulum, a cytoplasm, or a nucleus. Item 20. The method according to item 19.

21. The nucleic acid sequence is encoded by the RNA transcribed from the gene of interest within the intracellular target. The method of claim 19 or 20, wherein the binding occurs.

22. The gene of interest is a gene whose expression or overexpression is involved in the disease process.

22. The method of claim 21 , wherein the

23. The binding of the nucleic acid sequence to RNA transcribed from the gene of interest results in 23. The method of claim 21 or 22, which results in a decrease in expression of the target gene.

24. 15 to 17, wherein the MGS peptide is one or more of the MGS peptides listed in Table 1.

24. The method according to any one of claims 23.

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