CN117024557B - Application of 14-3-3 protein theta isomer as extracellular vesicle scaffold protein and extracellular vesicle - Google Patents

Application of 14-3-3 protein theta isomer as extracellular vesicle scaffold protein and extracellular vesicle Download PDF

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CN117024557B
CN117024557B CN202311301864.2A CN202311301864A CN117024557B CN 117024557 B CN117024557 B CN 117024557B CN 202311301864 A CN202311301864 A CN 202311301864A CN 117024557 B CN117024557 B CN 117024557B
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protein
theta
isomer
scaffold
exosomes
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CN117024557A (en
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葛啸虎
董凤伟
陆路
郭小飞
尹建新
陈宁
韩春乐
王淼
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Tianjin Exosome Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Abstract

The invention provides an application of a 14-3-3 protein theta isomer as an extracellular vesicle scaffold protein and an extracellular vesicle, and relates to the field of biotechnology. The 14-3-3 protein theta isomer or the fragment thereof provided by the invention is applied to the scaffold protein of the extracellular vesicles, the over-expression of the scaffold protein does not influence the growth of cells, and the enrichment degree of the scaffold protein in the extracellular vesicles is higher than that of the scaffold protein used for treatment in the prior art. The extracellular vesicles provided by the invention comprise a scaffold protein and a bioactive domain combined with the scaffold protein, wherein the scaffold protein is 14-3-3 protein theta isomer or a fragment thereof. The invention provides a novel scaffold protein, and provides a novel connection basis with higher abundance for the drug loading or marking application of extracellular vesicles.

Description

Application of 14-3-3 protein theta isomer as extracellular vesicle scaffold protein and extracellular vesicle
Technical Field
The invention relates to the technical field of biology, in particular to application of a 14-3-3 protein theta isomer as an extracellular vesicle scaffold protein and an extracellular vesicle.
Background
Adeno-associated virus (AAV) is currently considered the safest and most promising drug delivery vehicle due to its extremely low immunogenicity. But its small loading (less than 4.7 k) limits its partial application in the field of gene therapy. The exosomes belong to nano-sized particles, have low immunogenicity, and are suitable to be used as a drug carrier. And the volume of the exosome is obviously larger than that of AAV, and the exosome can accommodate 6k nucleic acid and has certain advantages compared with AAV. In the natural exosome, some proteins can be remarkably enriched, and can be used as scaffold proteins, and after fusion expression of a specific protein and the scaffold proteins, the specific protein can also be enriched, so that loading of proteins and nucleic acids on the exosome is realized. Therapeutic effects are achieved by delivering exosomes to recipient cells, allowing the loaded proteins or nucleic acids to function.
Some scaffold proteins commonly used in the prior art, such as CD63, CD81, etc., are not evenly distributed in the exosomes, only a portion of the exosomes contain such proteins. In drug delivery, the defects of the scaffold protein can influence the exosome delivery efficiency, thereby limiting the potential clinical application value of the engineering exosome. There is therefore a need to find new scaffold proteins to improve the current state of exosome scaffold proteins.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the application of 14-3-3 protein theta isomer or fragments thereof as extracellular vesicle scaffold protein, wherein the 14-3-3 protein theta isomer or fragments thereof is used as the extracellular vesicle scaffold protein, the extracellular vesicle is not influenced by the over-expression, the enrichment degree in the extracellular vesicle is higher than that of the scaffold protein for the existing treatment, and the distribution of the screened scaffold protein in the extracellular vesicle is at least equal to that of the commonly used scaffold protein (such as CD 63).
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the present invention there is provided the use of the 14-3-3 protein theta isomer or fragment thereof as an extracellular vesicle scaffold protein.
In some alternative embodiments, the 14-3-3 protein theta isomer has at least the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 6;
or the 14-3-3 protein theta isomer has an amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence with at least 85% sequence identity with SEQ ID NO. 1.
In some alternative embodiments, the extracellular vesicles include one or more of exosomes, microvesicles, apoptotic bodies, tumor vesicles, and nanovesicles;
and/or, the extracellular vesicles are extracellular vesicles that are localized to hepatocytes;
and/or, the 14-3-3 protein theta isomer or fragment thereof is localized to extracellular vesicles.
In some alternative embodiments, the 14-3-3 protein theta isoform or fragment thereof is overexpressed in cells that produce said extracellular vesicles.
According to another aspect of the present invention, there is also provided an extracellular vesicle comprising a scaffold protein and an active domain; the scaffold protein comprises a 14-3-3 protein theta isomer or fragment thereof.
In some alternative embodiments, the 14-3-3 protein theta isomer has at least the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 6;
or the 14-3-3 protein theta isomer has an amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence with at least 85% sequence identity with SEQ ID NO. 1.
In some alternative embodiments, the active domain comprises a polypeptide, polynucleotide, compound, or any combination thereof.
In some alternative embodiments, the scaffold protein is directly linked to the active domain or linked to the active domain through a linker.
In some alternative embodiments, the active domain comprises any one or a combination of the following (i) - (vi):
antisense oligonucleotides, mRNA, siRNA, miRNA, shRNA, lncRNA, circRNA or any combination thereof;
(ii) an antibody or antigen-binding fragment thereof, including a full-length antibody, scFv, (scFv) 2 、Fab、Fab'、F（ab'） 2 、F（ab1） 2 Fv, dAb and Fd fragments, bifunctional antibodies, antibody-related polypeptides or any fragment thereof or any combination thereof;
(iii) a nucleotide binding protein or fragment thereof;
(iv) a nucleotide binding protein or fragment thereof, and the nucleotide binding protein or fragment thereof binds to a polynucleotide;
(v) fluorescent marker proteins and/or tag proteins;
(vi) combinations of one or more of targeting peptides, recombinant peptides and therapeutic peptides.
In some alternative embodiments, the nucleotide binding protein is selected from the group consisting of Ku protein, sm7 protein, MS2 coat protein, PP7 coat protein, com RNA binding protein, or aptamer ligand, or any combination, functional variant, fragment, or domain thereof.
According to another aspect of the present invention, there is also provided an expression system comprising:
a first expression element encoding a scaffold protein, wherein the scaffold protein is a 14-3-3 protein theta isomer or fragment thereof;
and a second expression element encoding an active domain.
According to another aspect of the present invention there is also provided a cell comprising the expression system described above.
According to another aspect of the present invention, there is also provided a method for preparing an extracellular vesicle, comprising overexpressing a polypeptide comprising a 14-3-3 protein theta isomer or a fragment thereof in a cell, and isolating or producing the extracellular vesicle from the cell or a culture system comprising the cell.
According to another aspect of the present invention, there is also provided the use of an extracellular vesicle as described above, or an expression system as described above, or a cell as described above, or a method of preparation of an extracellular vesicle as described above, in the manufacture of a medicament for use in the prevention or treatment of a disease; such diseases include cancer, inflammatory disorders, neurodegenerative disorders, central nervous diseases or metabolic diseases.
According to another aspect of the present invention, there is also provided a pharmaceutical composition comprising the extracellular vesicles described above or extracellular vesicles prepared by the method of preparing extracellular vesicles described above; and a pharmaceutically acceptable carrier.
Compared with the prior art, the invention has the following beneficial effects:
the application of the 14-3-3 protein theta isomer or the fragment thereof provided by the invention as the extracellular vesicle scaffold protein provides a new connection basis with higher abundance for the drug loading or marking application of extracellular vesicles. The distribution of the 14-3-3 protein theta isomer or fragment thereof as a scaffold protein in extracellular vesicles can at least reach the level of a scaffold protein commonly used, such as CD 63. The 14-3-3 protein theta isomer or fragment thereof can be enriched on exosomes and can be used as exosome scaffold proteins to carry exogenous proteins or nucleic acids to achieve the desired effect. The extracellular vesicles provided by the invention comprise 14-3-3 protein theta isomers or fragments thereof, and the 14-3-3 protein theta isomers are used as protein scaffolds to load other active structural domains, such as polypeptides, polynucleotides and the like, so that the functions of exosomes are enriched, and the extracellular vesicles can be used as drug carriers and have tracing and targeting functions, and further can be used for preparing drugs so as to improve the therapeutic effect and targeting capability of the drugs.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows TEM results of exosomes overexpressing different scaffold proteins in example 2;
FIG. 2 shows the results of a marker immunoblot assay for exosomes overexpressing different scaffold proteins in example 2, wherein lane 1 is 293 cell lysate, lane 2 is 293Evs, lane 3 is YWHAQ-293Evs, lane 4 is CD63-293Evs, lane 5 is MFGE8-293Evs, lane 6 is PTGFRN-293Evs, and lane 7 is Basp-1-293Evs;
FIG. 3 shows the results of HPLC purity characterization after purification of different exosomes in example 2, wherein a is 293Evs, b is YWHAQ-Evs, c is CD63-293Evs;
FIG. 4 shows the results of HPLC purity characterization after purification of different exosomes in example 2, wherein a is MFGE8-Evs, b is PTGFRN-Evs, c is Basp-1-293Evs;
FIG. 5 is a graph showing the characterization of the scaffold distribution ratio of different exosomes in example 3;
FIG. 6 is a graph showing the results of characterization of scaffold abundance for different exosomes in example 3;
FIG. 7 shows the characterization of the purity (a), morphology (b), particle size (c) and loaded EGFP protein (d) of the exosomes overexpressing the truncated-YWHAQ scaffold protein in example 4;
FIG. 8 shows the characterization results of purity (a), particle size (b) and morphology (c) of exosomes YWHAQ-MCP-MS2-FIX-Fc 293Evs prepared in example 5;
FIG. 9 shows the characterization results of purity (a), particle size (b) and morphology (C) of exosomes YWHAQ-L7 Ae-C/Dbox-FIX-Fc 293Evs prepared in example 5;
FIG. 10 shows the results of characterization of purity (a), particle size (b) and morphology (c) of exosomes YWHAQ-FIX-Fc 293Evs prepared in example 5;
FIG. 11 shows the results of characterization of purity (a), particle size (b) and morphology (c) of exosomes YWHAQ-293Evs prepared in example 5;
FIG. 12 is a graph showing the results of the characterization of mRNA loading of YWHAQ fusion different RNA binding proteins in example 5;
FIG. 13 shows the results of characterization of purity (a), particle size (b), antibody positive rate (c) and morphology (d) of exosome CD19scfv-YWHAQ-EGFP 293Evs prepared in example 6;
FIG. 14 shows the results of characterization of purity (a), particle size (b), antibody positive rate (c) and morphology (d) of exosomes YWHAQ-EGFP 293Evs prepared in example 6;
FIG. 15 shows the characterization results of purity (a), particle size (b), antibody positive rate (c) and morphology (d) of the prepped ypaq scaffold prepped by example 6 that did not overexpress the YWHAQ scaffold;
FIG. 16 shows the results of the Elisa assay for binding of CD19scfv to CD19 antigen on the surface of the exosome in example 6;
FIG. 17 shows the results of characterization of the exosome purity (a), particle size (b), targeting peptide positive ratio (c) and morphology (d) of the exosome gp17-YWHAQ-EGFP-Flag 293Evs prepared in example 6;
FIG. 18 shows the results of characterization of exosome purity (a), particle size (b), targeting peptide positive ratio (c) and morphology (d) of exosome YWHAQ-EGFP-Flag 293Evs prepared in example 6;
FIG. 19 shows characterization results of the purity (a), particle size (b), targeting peptide positive ratio (c) and morphology (d) of the epi 293F cell exosome 293Evs not overexpressing YWHAQ scaffolds prepared in example 6;
FIG. 20 is a graph showing the results of phagocytic distribution of targeted peptide exosomes in liver organoids in example 6, with the ordinate indicating the overlapping area of the exosomes carrying Flag tag and hepatocyte surface tag DPPIV co-staining;
FIG. 21 is an effect of exosomes targeting peptides on in vivo distribution in exosome mice in example 7.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.
Term interpretation:
unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.
The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some alternative embodiments, the term "about" means a range of ±10% of the numerical value following that. In some alternative embodiments, the term "about" means a range of ±5% of the numerical value thereafter.
The term "substitution" or "substitution" of an amino acid as used herein may refer to a substitution of a conservative amino acid, wherein the amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid residue families having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such substitution is considered conservative. In another aspect, a string of amino acids may be conservatively substituted with a structurally similar string that differs in the order and/or composition of the side chain family members.
"percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since the gaps are not nucleotides or amino acids, the gaps present in the target sequence are not taken into account. Also, since the target sequence nucleotide or amino acid is counted, and the nucleotide or amino acid from the reference sequence is not counted, gaps in the reference sequence are not counted.
Percent sequence identity can be calculated by the following procedure: determining the number of positions in which the same amino acid residue or nucleobase occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software that is readily available for online use and download. Suitable software programs are available from a variety of sources for alignment of protein and nucleotide sequences. One suitable program for determining the percent sequence identity is the bl2seq, which is part of the BLAST suite of programs available from the national center for Biotechnology information BLAST website (BLAST. Ncbi. Lm. Nih. Gov). Bl2seq uses BLASTN or BLASTP algorithms to make a comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, needle, stretcher, water or a part of the Matcher, bioinformatics program EMBOSS kit, and are also available from European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.
The term "extracellular vesicle", "outer vesicle" or "EV" as used herein refers to a cell-derived vesicle comprising a membrane encapsulating an interior space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, microvesicles, apoptotic bodies, tumor vesicles, nanovesicles, etc.) that have a diameter that is smaller than the diameter of the cells from which they are derived. In some aspects, the extracellular vesicles have diameters in the range of 20nm to 1000nm, and can comprise various macromolecular payloads within the interior space (i.e., lumen), displayed on the outer surface of the extracellular vesicles, and/or across the membrane. In some aspects, the payload may include a nucleic acid, a protein, a carbohydrate, a lipid, a small molecule, and/or a combination thereof. In certain aspects, the extracellular vehicle comprises a scaffold moiety. By way of example and not limitation, extracellular vesicles include apoptotic bodies, cell debris, cell-derived vesicles obtained by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solutions), vesicular organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosomal fusion with plasma membrane). Extracellular vesicles may be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells and/or cultured cells. In some aspects, the extracellular vesicles are produced by cells that express one or more transgene products.
The term "exosomes" as used herein refers to extracellular vesicles having a diameter between 20-300 nm (e.g. between 40-200 nm). Exosomes comprise membranes that encapsulate an internal space, and in some aspects, may be produced by cells by direct plasma membrane budding or by late endosomes or fusion of the polycystic body with the plasma membrane. In certain aspects, the exosomes comprise a scaffold moiety. As described below, exosomes may be derived from producer cells and isolated from producer cells according to their size, density, biochemical parameters, or a combination thereof. In some aspects, an EV (e.g., exosome) of the present disclosure is produced by a cell expressing one or more transgene products.
The term "nanovesicle" as used herein refers to extracellular vesicles having a diameter between 20-250 nm (e.g., between 30-150 nm) and being produced by a cell (e.g., a producer cell) by direct or indirect manipulation such that the cell would not produce nanovesicles without manipulation. Suitable manipulations of the cells for the production of nanovesicles include, but are not limited to, continuous extrusion, treatment with alkaline solutions, sonication, or combinations thereof. In some aspects, the population of nanovesicles described herein is substantially free of vesicles derived from cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. In certain aspects, the nanocapsules comprise a scaffold moiety, such as scaffold X and/or scaffold Y. The nanovesicles, once derived from the producer cell, may be isolated from the producer cell based on their size, density, biochemical parameters, or a combination thereof.
The term "active domain" or "bioactive domain" as used herein is used interchangeably to refer to any molecule that can be linked to a scaffold protein through an anchoring moiety, wherein the molecule can have therapeutic or prophylactic effect in a subject in need thereof, or can be used for diagnostic purposes. Thus, for example, the term bioactive domain includes a protein (e.g., an antibody, a protein, a polypeptide, and derivatives, fragments, and variants thereof), a nucleotide binding protein, a polynucleotide, or a chemical compound.
The term "antibody" as used herein encompasses immunoglobulins (whether naturally occurring or partially or fully synthetically produced) and fragments thereof. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain. An "antibody" also includes polypeptides that comprise a framework region from an immunoglobulin gene or fragment thereof that specifically binds and recognizes an antigen. The use of the term antibody is intended to include whole antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, fragments thereof, and also includes single chain antibodies, human sourcesAntibodies, murine antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments (such as, for example, scFv, (scFv) 2 Fab, fab 'and F (ab') 2 、F（ab1） 2 Fv, dAb and Fd fragments), bifunctional antibodies and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.
The term "polynucleotide" as used herein refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes three-stranded, double-stranded and single-stranded deoxyribonucleic acid ("DNA"), as well as three-stranded, double-stranded and single-stranded ribonucleic acid ("RNA"). It also includes modified (e.g., by alkylation and/or by capping) and unmodified forms of the polynucleotide. More particularly, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose); polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or non-spliced; any other type of polynucleotide that is an N-or C-glycoside of a purine or pyrimidine base; and other polymers containing an oligonucleotide backbone, such as polyamides (e.g., peptide nucleic acids "PNAs") and polymorpholino polymers; and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that permits base pairing and base stacking, such as found in DNA and RNA.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. Such polymers include gene products, naturally occurring polymers, synthetic polymers, homologs, orthologs, fragments and other equivalents, variants and analogs of the foregoing. The polymer may comprise modified amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation to a labeling component. Also included in the definition are polypeptides containing, for example, one or more amino acid analogs (including, for example, unnatural amino acids, such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. The polymer may be a single molecule or may be a multi-molecular complex, such as a dimer, trimer or tetramer. They may also comprise single-or multi-chain polypeptides. Most commonly, disulfide bonds are present in multi-chain polypeptides. The term polypeptide may also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acid. In some aspects, a "peptide" may be less than or equal to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
The term "fusion protein" as used herein refers to a protein in which two or more proteins or fragments thereof are co-linearly linked by respective peptide backbones using genetic expression of the polynucleotide encoding the protein or protein synthesis methods.
As used herein, the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. A linker links or genetically fuses a first linear amino acid sequence to a second linear amino acid sequence, which is not naturally linked or genetically fused to the second linear amino acid sequence in nature.
The linker of any of the embodiments herein is implemented according to any of the following embodiments:
in some alternative embodiments, the linker is a polypeptide linker and/or a non-polypeptide linker.
In some alternative embodiments, the linker may be a (chemical) covalent linkage.
In some alternative embodiments, the polypeptide linker may comprise at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids. In some alternative embodiments, the linker comprises one or more amino acids. In some alternative embodiments, the linker comprises a Gly-Ser (GS) linker. In some alternative embodiments, the GS linker comprises (G4S) n, where n is an integer between 1 and 10. In some alternative embodiments, the GS linker comprises (G3S) n, where n is an integer between 1 and 10.
In some alternative embodiments, the polypeptide linker is synthetic, i.e., non-naturally occurring, which is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation such as an addition, substitution, or deletion).
In some alternative embodiments, the linker may be easily cleaved ("cleavable linker") to facilitate release of the exogenous bioactive moiety. In some alternative embodiments, the linker is a "reduction-sensitive linker". In some alternative embodiments, the reduction-sensitive linker contains a disulfide bond. In some alternative embodiments, the linker is an "acid labile linker". In some alternative embodiments, the acid labile linker contains hydrazone. Suitable acid labile linkers also include, for example, cis aconitic acid linkers, hydrazide linkers, thiocarbamoyl linkers, or any combination thereof. In some alternative embodiments, the ASO is associated with an EV (e.g., exosome) by means of a linker. In some alternative embodiments, the linker comprises an acrylic phosphoramidite (e.g., ACRYDITETM), adenylation, azide (NHS Ester), digoxin (NHS Ester), cholesterol-TEG, I-LINKERTM, an amino modifier (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, or Uni-LinkTM amino modifier), alkyne, 5' hexynyl, 5-octadiynyl dU, biotinylation (e.g., biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, or desthiobiotin), thiol modification (thiol modifier C3S-S, dithiol, or thiol modifier C6S-S), or any combination thereof.
In some alternative embodiments, the linker comprises a terpene, such as nerolidol, farnesol, limonene, linalool, geraniol, carvone, cumarone, or menthol; lipids such as palmitic acid or myristic acid; cholesterol; oil-based; a retinyl group; a cholesterol residue; cholic acid; adamantane acetic acid; 1-pyrenebutyric acid; dihydrotestosterone; 1, 3-bis-O (hexadecyl) glycerol; geranyloxyhexyl; hexadecyl glycerol; borneol; 1, 3-propanediol; heptadecyl; o3- (oleoyl) lithocholic acid; o3- (oleoyl) cholic acid; dimethoxytrityl group; phenoxazine, maleimide moiety, glucuronidase type, CL2A-SN38 type, folic acid; a carbohydrate; vitamin A; vitamin E; vitamin K, or any combination thereof. In certain aspects, the ASO comprises a cholesterol tag, and the cholesterol tag is associated with a membrane of an EV (e.g., exosome). In some alternative embodiments, the linker comprises a non-cleavable linker. In some alternative embodiments, the linker comprises tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), succinimide, or any combination thereof. In some alternative embodiments, the linker comprises a spacer unit that links the bioactive molecule to the linker. In some alternative embodiments, one or more linkers include smaller units (e.g., HEG, TEG, glycerol, C2 to C12 alkyl, etc.) linked together. In one aspect, the linkage is an ester linkage (e.g., a phosphodiester or phosphorothioate) or other linkage. In some alternative embodiments, the linker comprises polyethylene glycol (PEG), characterized by the formula R3- (O-CH 2-CH 2) n-or R3- (0-CH 2-CH 2) n-O-, wherein R3 is hydrogen, methyl or ethyl, and n has a value of 2 to 200. In some alternative embodiments, the linker comprises a spacer, wherein the spacer is PEG. In some alternative embodiments, the PEG linker is an oligomeric ethylene glycol, such as diethylene glycol, triethylene glycol, tetraethylene glycol (TEG), pentaethylene glycol, or hexaethylene glycol (HEG) linker.
In some embodiments, the linker may include a transmembrane peptide. In some embodiments, the scaffold protein binds to the active domain via a linker comprising a transmembrane peptide, thereby displaying the active domain within the lumen of an outer vesicle. In some embodiments, the scaffold protein binds to the nucleotide binding protein or fragment thereof via a transmembrane peptide to form a fusion protein. The nucleotide binding protein or fragment thereof is further bound to a polynucleotide sequence. In some embodiments, the transmembrane peptide may have, for example, the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 12 or an amino acid sequence having a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence of SEQ ID NO. 12.
In some alternative embodiments, two or more connectors may be connected in series. When there are multiple linkers, each linker may be the same or different. In general, the linker provides flexibility or prevents/improves steric hindrance. The linker is typically not cleaved; however, in certain aspects, such cutting may be desirable. Thus, in some alternative embodiments, the linker may comprise one or more protease cleavable sites that may flank the linker within or at either end of the linker sequence.
The term "preventing" as used herein refers to partially or completely delaying the onset of a disease, disorder, and/or condition; partially or completely delay the onset of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and/or condition; partially or completely delay the onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; partially or completely delay progression from a particular disease, disorder, and/or condition; and/or reduce the risk of developing a pathology associated with a disease, disorder, and/or condition.
The term "treatment" as used herein refers to, for example, a reduction in the severity of a disease or condition; shortening duration of disease course; improvement or elimination of one or more symptoms associated with the disease or condition; providing a beneficial effect to a subject suffering from a disease or condition, but not necessarily curing the disease or condition. The term also includes the prevention or prophylaxis of a disease or condition or symptoms thereof.
The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical formulation that is non-toxic to a subject other than the active ingredient.
The technical scheme of the invention is further described below in connection with the preferred embodiments.
The 14-3-3 protein theta isoform (also known as tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activator protein, theta polypeptide) is encoded by the human YWHAQ gene and plays an important role in controlling apoptosis by interacting with ASK1, c-jun nh terminal kinase and p38 Mitogen Activated Protein Kinase (MAPK). The inventors have unexpectedly found that the 14-3-3 protein theta isomer can be enriched on exosomes and can be used as exosome scaffold proteins to carry exogenous proteins or nucleic acids to achieve the desired effect.
Thus, in light of the above findings, the present invention provides the use of the 14-3-3 protein theta isomer or fragment thereof as an extracellular vesicle scaffold protein.
In some alternative embodiments, the 14-3-3 protein theta isomer or fragment thereof is localized to an extracellular vesicle. In some alternative embodiments, the 14-3-3 protein theta isomer or fragment thereof is localized on, within, or outside the membrane of an extracellular vesicle. In some alternative embodiments, the 14-3-3 protein theta isomer or fragment thereof is located within the lumen of an extracellular vesicle.
In some alternative embodiments, the extracellular vesicles include one or more of exosomes, microvesicles, apoptotic bodies, tumor vesicles, and nanovesicles.
In some alternative embodiments, the extracellular vesicles are extracellular vesicles that localize to hepatocytes.
In some alternative embodiments, the 14-3-3 protein theta isoform or fragment thereof is overexpressed in cells producing the extracellular vesicles, such that the extracellular vesicles obtain the 14-3-3 protein theta isoform or fragment thereof as a scaffold protein.
In some alternative embodiments, the amino acid sequence of the 14-3-3 protein theta isomer is of human origin.
In some alternative embodiments, the 14-3-3 protein theta isomer or fragment thereof as a scaffold protein may also be engineered, such as by truncation, mutation, insertion of exogenous sequences, or modification.
In some alternative embodiments, the 14-3-3 protein theta isomer has at least the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 6.
In some alternative embodiments, the 14-3-3 protein theta isomer has the amino acid sequence set forth in SEQ ID NO. 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 1. The amino acid sequence shown in SEQ ID NO. 1 is derived from human.
In some alternative embodiments, the amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 1 or to SEQ ID NO. 6 is of human origin.
In some alternative embodiments, the amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 1 or to SEQ ID NO. 6 has the same function as SEQ ID NO. 1 or to SEQ ID NO. 6, including localization to extracellular vesicles and display of the active domain on the outer surface of an outer vesicle or within the lumen of an outer vesicle.
In some alternative embodiments, the 14-3-3 protein theta isomer or fragment thereof as a scaffold protein is also fused directly or via a linker to other domains fused to the N-terminus and/or the carbon-terminus of the 14-3-3 protein theta isomer or fragment thereof, such as may be, but not limited to, the active domains in any of the embodiments herein.
In some alternative embodiments, the scaffold protein is fused at the N-terminus to a short peptide chain capable of expressing a targeting peptide, recombinant peptide, therapeutic peptide, or linker.
In other embodiments, the scaffold protein is fused at the C-terminus to a short peptide chain capable of expressing a therapeutic peptide, recombinant peptide, or linker.
In some embodiments, the scaffold protein C-terminal fusion RNA binding protein is capable of binding to RNA containing a specific domain, the RNA containing a specific domain comprising a functional region having a pharmaceutical effect and a linking region for linking the RNA binding protein; the RNA used in the treatment may be mRNA, miRNA, lncRNA, circRNA or siRNA.
According to another aspect of the present invention, there is provided an extracellular vesicle comprising a scaffold protein and an active domain; the scaffold protein comprises a 14-3-3 protein theta isomer or fragment thereof.
In some alternative embodiments, the amino acid sequence of the 14-3-3 protein theta isomer is of human origin.
In some alternative embodiments, the active domain may be displayed on the outer surface of the outer vesicle or within the lumen of the outer vesicle by a scaffold protein 14-3-3 protein theta isomer of the present disclosure, or a fragment thereof.
In some alternative embodiments, the 14-3-3 protein theta isomer has at least the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 6.
In some alternative embodiments, the 14-3-3 protein theta isomer has the amino acid sequence set forth in SEQ ID NO. 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 1.
In some alternative embodiments, the amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 1 or to SEQ ID NO. 6 is of human origin.
In some alternative embodiments, the amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 1 or to SEQ ID NO. 6 has the same function as SEQ ID NO. 1 or to SEQ ID NO. 6, including localization to extracellular vesicles and display of the active domain on the outer surface of an outer vesicle or within the lumen of an outer vesicle.
In some alternative embodiments, the active domain comprises a polypeptide, polynucleotide, compound, or any combination thereof.
In some alternative embodiments, the polypeptides include, but are not limited to, one or more of antibodies or antigen binding fragments thereof, nucleotide binding proteins or fragments thereof, fluorescent-tagged proteins, polypeptides having therapeutic and/or targeting effects.
In some alternative embodiments, the scaffold protein binds to the active domain directly or through a linker.
In some alternative embodiments, the active domain comprises one or a combination of the following (i) - (vi):
one or a combination of several of (i) polynucleotides including, but not limited to, antisense oligonucleotides (ASOs), mRNA, siRNA, miRNA, shRNA, lncRNA, circRNA or any combination thereof;
(ii) an antibody or antigen-binding fragment thereof, or any combination thereof, including a full-length antibody, scFv, (scFv) 2 、Fab、Fab'、F（ab'） 2 、F（ab1） 2 Fv, dAb and Fd fragments, bifunctional antibodies, antibody-related polypeptides or any fragment thereof or a combination of any fragment thereof;
(iii) a nucleotide binding protein or fragment thereof;
(iv) a nucleotide binding protein or fragment thereof, and the nucleotide binding protein or fragment thereof binds to a polynucleotide;
(v) fluorescent marker proteins and/or tag proteins;
(vi) combinations of one or more of targeting peptides, recombinant peptides and therapeutic peptides.
In some alternative embodiments, the nucleotide binding protein of (iii) or (iv) is selected from the group consisting of Ku protein, sm7 protein, MS2 coat protein, PP7 coat protein, com RNA binding protein, or aptamer ligand, or any combination, functional variant, fragment, or domain thereof.
In some alternative embodiments, (iii) or (iv) the scaffold protein forms a fusion protein with a nucleotide binding protein or fragment thereof.
In some alternative embodiments, the extracellular vesicles are exosomes, the amino acid sequence of the 14-3-3 protein theta isomer is shown in SEQ ID No. 1, the active domain is a nucleotide binding protein, the nucleotide binding protein is archaebacteria L7Ae, and the amino acid sequence is shown in SEQ ID No. 8.
In some alternative embodiments, the extracellular vesicles are exosomes, the amino acid sequence of the 14-3-3 protein theta isomer is shown in SEQ ID No. 1, the active domain is a nucleotide binding protein, the nucleotide binding protein is an RNA binding protein MCP protein of MS2 phage, and the amino acid sequence is shown in SEQ ID No. 7.
In some alternative embodiments, the extracellular vesicles are exosomes, the amino acid sequence of the 14-3-3 protein theta isomer is shown in SEQ ID No. 1, and the active domain is an scFv (single chain antibody). In some alternative embodiments, the scFv is derived from an anti-CD 19 antibody.
In some alternative embodiments, the extracellular vesicles are exosomes, the amino acid sequence of the 14-3-3 protein theta isoform is shown in SEQ ID No. 1, and the active domain is a short peptide derived from phage P17 protein, which targets hepatocytes.
In some alternative embodiments, the active domain further comprises a fluorescent protein, such as EGFP.
According to another aspect of the present invention, there is also provided an expression system comprising: a first expression element encoding a scaffold protein, wherein the scaffold protein is a 14-3-3 protein theta isomer or fragment thereof; and a second expression element encoding an active domain.
In some alternative embodiments, the 14-3-3 protein theta isomer has at least the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 6.
In some alternative embodiments, the 14-3-3 protein theta isomer has the amino acid sequence set forth in SEQ ID NO. 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 1.
In some alternative embodiments, the amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 1 or to SEQ ID NO. 6 is of human origin.
In some alternative embodiments, the amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 1 or to SEQ ID NO. 6 has the same function as SEQ ID NO. 1 or to SEQ ID NO. 6, including localization to extracellular vesicles and display of the active domain on the outer surface of an outer vesicle or within the lumen of an outer vesicle.
In some alternative embodiments, the active domain comprises a polypeptide, polynucleotide, compound, or any combination thereof.
In some alternative embodiments, the polypeptides include, but are not limited to, one or more of antibodies or antigen binding fragments thereof, nucleotide binding proteins or fragments thereof, fluorescent-tagged proteins, polypeptides having therapeutic and/or targeting effects.
In some alternative embodiments, the scaffold protein binds to the active domain directly or through a linker.
In some alternative embodiments, the active domain comprises one or a combination of the following (i) - (vi):
one or a combination of several of (i) polynucleotides including, but not limited to, antisense oligonucleotides (ASOs), mRNA, siRNA, miRNA, shRNA, lncRNA, circRNA or any combination thereof;
(ii) an antibody or antigen-binding fragment thereof, or any combination thereof, including a full-length antibody, scFv, (scFv) 2 、Fab、Fab'、F（ab'） 2 、F（ab1） 2 Fv, dAb and Fd fragments, bifunctional antibodies, antibody-related polypeptides or any fragment thereof or a combination of any fragment thereof;
(iii) a nucleotide binding protein or fragment thereof;
(iv) a nucleotide binding protein or fragment thereof, and the nucleotide binding protein or fragment thereof binds to a polynucleotide;
(v) fluorescent marker proteins and/or tag proteins;
(vi) combinations of one or more of targeting peptides, recombinant peptides and therapeutic peptides.
In some alternative embodiments, the nucleotide binding protein of (iii) or (iv) is selected from the group consisting of Ku protein, sm7 protein, MS2 coat protein, PP7 coat protein, com RNA binding protein, or aptamer ligand, or any combination, functional variant, fragment, or domain thereof.
In some alternative embodiments, (iii) or (iv) the scaffold protein forms a fusion protein with a nucleotide binding protein or fragment thereof.
In some alternative embodiments, the expression system may be selected from a prokaryotic expression system, a eukaryotic expression system, or a viral expression system. In some alternative embodiments, the expression system may include a prokaryotic vector, eukaryotic vector, viral vector, or the like.
According to another aspect of the present invention there is also provided a cell comprising the expression system described above.
In some alternative embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some alternative embodiments, the prokaryotic cell may be selected from E.coli or B.subtilis, etc., e.g., E.coli BL21, T7E, C41, or Arctic, etc. In some alternative embodiments, the eukaryotic cell may be selected from a yeast cell, an insect cell, a plant cell, or a mammalian cell, etc., such as a yeast cell, CHO cell, 293 cell, vero cell, NSO cell, etc.
According to another aspect of the present invention, there is also provided a method for preparing an extracellular vesicle, which comprises overexpressing a polypeptide comprising the 14-3-3 protein theta isoform or a fragment thereof in a cell, and isolating or producing the extracellular vesicle from the cell or a culture system comprising the cell. The skilled artisan can select any one or more of the optional, conventional methods of separation or manufacture in the art, such as centrifugation, continuous extrusion, treatment with alkaline solution, and sonication, as described herein, depending on the biological characteristics of the extracellular vesicles to be obtained. Those skilled in the art will be able to isolate or produce extracellular vesicles of interest from cells or cell culture systems according to the general knowledge in the art, and the invention is not limited to a particular isolation method.
In some alternative embodiments, the cell that overexpresses the 14-3-3 protein theta isoform or fragment thereof is a cell of the expression system of the preceding embodiments comprising the first expression element and the second expression element.
In some alternative embodiments, the method of preparation is a method of preparation of exosomes.
In some alternative embodiments, the method of preparing an exosome comprises isolating the exosome from a cell culture system.
According to another aspect of the present invention, there is also provided a use of the extracellular vesicles of the preceding embodiment, or the expression system of the preceding embodiment, or the cells of the preceding embodiment, or the method of preparation of the extracellular vesicles of the preceding embodiment, in the manufacture of a medicament for the prevention or treatment of a disease; such diseases include cancer, inflammatory disorders, neurodegenerative disorders, central nervous diseases or metabolic diseases.
In some alternative embodiments, the extracellular vesicles are administered via intravenous, intraperitoneal, nasal, oral, intramuscular, subcutaneous, parenteral, or intratumoral administration.
In some alternative embodiments, the extracellular vesicles may be used for drug loading and/or marker loading.
According to another aspect of the present invention, there is also provided a pharmaceutical composition comprising the extracellular vesicles of the foregoing embodiment or the extracellular vesicles prepared by the method of preparing the extracellular vesicles of the foregoing embodiment; and a pharmaceutically acceptable carrier.
In some alternative embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, one or a combination of several of a buffer, excipient, stabilizer, or preservative.
In some alternative embodiments, the pharmaceutical composition is in the form of a tablet, powder, granule, pill, injection, suspension, powder, emulsion, aerosol, gel, eye drop, sustained release formulation, or sustained release implant. In some alternative embodiments, the pharmaceutical composition may be formulated as an injectable formulation. In some alternative embodiments, the formulation is suitable for intravitreal injection, subcutaneous, intradermal, intramuscular, intravenous, intrathecal or intrathecal administration.
In some alternative embodiments, the extracellular vesicles in the aforementioned medicament for the preparation of a medicament for the prevention or treatment of a disease or the extracellular vesicles in the aforementioned pharmaceutical composition are exosomes. Exosomes are rich in RNA (miRNA, lncRNA, circRNA, etc.), DNA, proteins and lipids, involved in intercellular molecular transfer. Exosomes are a highly heterogeneous population, including size heterogeneity, heterogeneity of content, functional heterogeneity, and source heterogeneity. Therefore, the exosomes produced by different cells have different molecular species and numbers, and the exosomes separated by different exosome purification modes have different molecular species and numbers. Exosomes are rich in various molecules, and when taken up by cells, can cause different degree influence to recipient cells, and natural exosomes that originate from different cells can have some functions of source cells, through reforming the cells, and then influence exosomes's function, are used for treatment or drug delivery.
The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.
Example 1 construction of expression vector
The expression plasmids pcDNA3.1 (+) -scaffold protein-EGFP-nanonuc were constructed from the YWHAQ-encoded 14-3-3 protein theta isomer (SEQ ID NO: 1) and the control scaffold proteins BASP-1 (SEQ ID NO: 2), PTGFRN (SEQ ID NO: 3), CD63 (SEQ ID NO: 4), MFGE8 (SEQ ID NO: 5), respectively. In the examples that follow, the gene YWHAQ, when named, to express the 14-3-3 protein theta isomer refers to the 14-3-3 protein theta isomer fraction in vectors, exosomes and cells, unless specifically indicated.
Example 2 preparation and validation of exosomes
Exosomes expressing EGFP-Nanoluc fusion expressed proteins with scaffolds YWHAQ, BASP-1, PTGFRN, CD63 and MFGE8 were prepared separately (named YWHAQ-293Evs, BASP-1-293Evs, PTGFRN-293Evs, CD63-293Evs, MFGE8-293Evs and 293 Evs).
2.1 Passage of 293s cells
Expi 293F cell resuscitating density is 3-10×10 5 The cell density is controlled to be 0.5-5 multiplied by 10 in each cell/ml in the passage period 6 Individual cells/ml.
2.2 Cell transfection
The passage cells in the step 2.1 are adjusted to be 1-3 multiplied by 10 before transfection 6 The concentration of each cell/ml and the concentration of the plasmid are 1-1.5 mug/ml, and the concentration ratio of the Polyethyleneimine (PEI) to the plasmid is controlled to be (1:1) - (4:1). The 5 plasmids obtained in example 1 were transfected into 293s cells under the conditions described above, and the proteins YWHAQ, BASP-1, PTGFRN, CD63 and MFGE8 were overexpressed in the cells. 37 ℃ 5% CO 2 Culturing for 48-72 h, and collecting cell culture supernatant for downstream purification.
2.3 exosome purification
Using the cell culture supernatant collected in step 2.2, ultrafiltering with 300KD membrane package, ultracentrifugation for 100000g×2h, molecular sieve filtration with 30-80nm aperture, ultracentrifugation for 100000g×2h, concentrating the sample, and finally obtaining exosome sample for nanoflow analysis.
2.4 exocrine physical examination
The purified exosomes are characterized, the morphological (TEM) characterization result is shown in figure 1, and the purified product is a typical exosome dish-shaped sinus membrane layer structure.
In addition, the expression of exosome markers CD81, TSG101, and golgi marker GM130 and endoplasmic reticulum marker Calexin from each group of cells was detected by Western Blot (Western Blot), and the results are shown in fig. 2. The characterization results of the purity (HPLC) are shown in FIG. 3-FIG. 4. As can be seen from fig. 2 to fig. 4, the obtained exosomes derived from each group of cells highly expressed CD81 and TSG101, while no expression of GM130 and Calexin was detected, further indicating that the obtained exosomes were obtained and had higher purity.
Example 3 distribution and abundance characterization of exosomes engineered with novel scaffold proteins on exosomes
3.1 comparison of exosome scaffold distribution characterization
The ratio of EGFP-positive particles in 20. Mu.L exosomes of the different scaffold proteins and negative control groups, respectively, was detected using Nano-flow, as shown in FIG. 5. Figure 5 shows that the proportion of YWHAQ positive exosome subpopulations in the overall exosome distribution is above 50% and higher than the currently disclosed exosome universal stent CD63.
3.2 comparison of exosome scaffold abundance characterization
The Nano-glo luciferase reagent is used for respectively detecting the Nanoluc enzyme activities in the exosomes with the same particle number of different scaffold proteins and the negative control group so as to characterize the abundance content of the fusion expression scaffold in the exosomes with the same particle number, as shown in fig. 6. Figure 6 shows that the abundance of ywaq scaffolds is greater than CD63 scaffolds.
Example 4: novel scaffold protein truncated protein for realizing exosome localization and loading foreign substances
In order to determine the shortest sequence for the 14-3-3 protein theta isomer to realize exogenous material loading, this example finally determines a structural domain according to the protein structure division of the 14-3-3 protein theta isomer, namely amino acids 1-67 of the sequence shown in SEQ ID NO:1, and names truncated 14-3-3 protein theta isomer structural domain (SEQ ID NO: 6) as the shortest sequence for realizing exosome localization loading.
The distribution and abundance in exosomes are verified as follows.
4.1 Construction and preparation of expression vector
(1.1) vector construction
And (3) fusing and expressing the C end of the truncated-YWHAQ and EGFP-Nanoluc, and constructing a verification expression vector pcDNA3.1-truncated-YWHAQ-EGFP-Nanoluc.
(1.2) plasmid preparation
Plasmid preparation was as in example 1.
(1.3) preparation of exosomes
The plasmid prepared in (1.2) was transfected into an Expi 293 cell, and the preparation, purification and quality control procedures were the same as in example 2. This was designated as truncated-YWHAQ-293 Evs.
4.2 EGFP is loaded into exogenesis experience evidence by truncated-YWHAQ-293Evs
Characterization of the high purity exosomes prepared in step (1.3) above, fig. 7 shows that the exosomes purity is approximately 100%, the morphology is of exosome dish-shaped sinus membrane layer structure, and the particle size distribution range is of exosome normal range. The positive rate of EGFP in exosomes is about 19.1%, which indicates that the truncated-YWHAQ can load EGFP into exosomes, and the exosome positioning and loading functions are reserved.
Example 5: YWHAQ as scaffold protein and fusion of RNA binding protein for mRNA loading
The RNA binding proteins selected in this example were the RNA binding protein MCP protein of MS2 phage (SEQ ID NO: 7) and the L7Ae of archaea (SEQ ID NO: 8), the recognition sequences of which were MS2 (SEQ ID NO: 9) and the C/D box sequence (SEQ ID NO: 10), respectively, and the mRNA selected was the mRNA encoded by the protein coagulation factor 9 and Fc fusion protein.
5.1 Construction and preparation of expression vector
(1.1) vector construction
1) Scaffold expression plasmid: the RNA binding proteins MCP and L7Ae are respectively fused to the C end of a 14-3-3 protein theta isomer (SEQ ID NO. 1), a GGGSlinker is used for connecting and respectively constructing a scaffold expression plasmid pcDNA3.1 (+) -YWHAQ-MCP/L7Ae (RNA binding protein), the 14-3-3 protein theta isomer and the RNA binding protein are fused and expressed, so that the 14-3-3 protein theta isomer is a scaffold capable of loading mRNA, and pcDNA3.1 (+) -YWHAQ is used as a control.
2) mRNA transcription plasmid: RNA binding protein recognition sequences (MCP recognizes MS2, L7Ae recognizes C/D box) are positioned at the 3' end of the loaded mRNA, and after the stop codon, mRNA transcription plasmid pcDNA3.1-FIX-Fc-MS2/C/Dbox (binding protein recognition sequence) is constructed before polyA, and pcDNA3.1-FIX-Fc is the corresponding plasmid with the control.
(1.2) plasmid preparation
Plasmid preparation was as in example 1.
5.2 Preparation of exosomes
The scaffold expression plasmid (pcDNA3.1 (+) -scaffold protein-MCP/L7 Ae) prepared in 5.1 was co-transfected with mRNA transcription plasmid (pcDNA3.1-FIX-Fc-MS 2/C/Dbox) to prepare mRNA-loaded exosomes. The preparation procedure was the same as in example 2, except that 4 sets of exosomes were prepared, each:
YWHAQ-MCP-MS2-FICX-Fc 293Evs: the exosomes prepared by co-transfecting pcDNA3.1 (+) -YWHAQ-MCP and pcDNA3.1-FIX-Fc-MS 2.
YWHAQ-L7Ae-C/D box-FIX-Fc 293Evs: the exosomes were prepared by co-transfecting pcDNA3.1 (+) -YWHAQ-L7Ae and pcDNA3.1-FIX-Fc-C/Dbox.
YWHAQ-FIX-Fc 293Evs: exosomes prepared by co-transfecting pcDNA3.1 (+) -YWHAQ and pcDNA3.1-FIX-Fc.
YWHAQ-293Evs: the exosomes prepared by transfecting pcDNA3.1 (+) -YWHAQ.
The analysis results of the morphology, particle size and purity of the 4 exosomes are shown in fig. 8 to 11. Fig. 8-11 show that the purity of the exosomes is close to 100%, the exosomes are in a dish-shaped sinus membrane layer structure, and the particle size distribution range is the normal range of the exosomes.
5.3 Exosome loading package target (FIX 9) mRNA characterization
Exosome sample FIX 9 mRNA calculation:
1) 200 μl of exosomes were taken to extract total RNA.
2) RT-qPCR was performed on RNA to detect the copy number of FIX mRNA in RNA.
3) The number of exosome particles concentration was measured for each sample.
4) The number of loaded copies of mRNA in the average individual exosomes was calculated.
As shown in FIG. 12, the exosomes YWHAQ-MCP-MS2-FICX-Fc 293Evs and YWHAQ-L7Ae-C/D box-FIX-Fc 293Evs fused to express the RNA binding protein scaffold can both promote the mRNA load compared with the YWHAQ-FIX-Fc 293Evs exosomes, so that after the scaffold protein is fused with the RNA binding protein, more mRNA can be wrapped into the exosomes to realize the mRNA loading energization of the scaffold protein.
Example 6: exosome targeting function
6.1 Exosome surface targeting antibody display
(1.1) construction of scaffold fusion expressed antibody plasmid
The 14-3-3 protein theta isomer is taken as an antibody display bracket, an expression vector is constructed, the exosome surface distributed by the chimeric bracket displays the antibody, the exosome targeting function is realized, and the displayed antibody is a human CD19 corresponding clone number FMC63 antibody. The plasmid was named pcDNA3.1-CD19scfv-YWHAQ-EGFP. Single chain antibodies (scFv) derived from FMC63 antibodies were fused to the N-terminus of the 14-3-3 protein theta isomer (SEQ ID NO. 1) and ligated using a transmembrane segment (SEQ ID NO. 12) and GGGSlinker.
(1.2) preparation of exosomes
Exosomes fusion expressing CD19scfv (designated CD19 scfv-ywaq-EGFP 293 Evs) and exosomes only overexpressing scaffold proteins (designated ywaq-EGFP 293 Evs) and 293F cell exosomes (designated 293 Evs) were prepared separately.
293s cells were passaged, transfected and exosome purified as in example 1.
(1.3) exocrine physical examination
Analyzing and detecting the morphology, particle size and purity of the sample obtained in the step (1.2) in the embodiment, wherein the distribution ratio of CD19scfv-YWHAQ-EGFP 293Evs in the prepared sample is about 38%, the particle size is 40-200 nm, the purity is more than 90%, and the morphology of the exosomes is an exosome dish-shaped sinus membrane structure when observed by an electron microscope as shown in the following figures 13-15.
(1.4) functional verification of CD19scfv-YWHAQ-EGFP 293Evs targeting CD19 antigen
1) The CD19 antigen is coated on a solid-phase carrier Elisa plate and is coated at 4 ℃ overnight;
2) After washing the plate with PBST buffer solution, 2% BSA was blocked at room temperature for 2h;
3) Incubating an equal amount of tested exosome samples after washing the plates, and standing for 1h at room temperature;
4) Incubating the following exosome surface marker CD9 antibody after washing, and then labeling secondary HRP antibody;
5) After the substrate is developed, the reaction is terminated and the absorbance of the sample is measured by OD 450. The test exosome samples were evaluated for CD19 antigen binding exosomes.
The results in fig. 16 show that exosome CD19scfv can specifically bind to CD19 antigen, thereby achieving exosome antigen targeting.
6.2 Exosome surface targeting peptide display
(2.1) construction of scaffold fusion expression targeting peptide plasmid
The exosome distribution protein 14-3-3 protein theta isomer is used as a target peptide display bracket, and the display target peptide is a short peptide (SEQ ID NO. 11) which targets liver cells and is derived from bacteriophage P17 protein, so that the exosome targeting liver cells are realized. The plasmid was designated pcDNA3.1-gp17-YWHAQ-EGFP. A short peptide derived from phage P17 protein was fused to the N-terminus of the 14-3-3 protein theta isomer (SEQ ID NO. 1) and ligated using a transmembrane segment (SEQ ID NO. 12) and GGGSlinker.
(2.2) preparation of exosomes
Preparation of fusion expressed gp17 targeting peptide exosomes (named gp17-YWHAQ-EGFP-Flag 293 Evs), exosomes not containing targeting peptide (named YWHAQ-EGFP-Flag 293 Evs) and 293 original exosomes (named 293 Evs), respectively
293s cells were passaged, transfected and exosome purified as in example 1.
(2.3) quality control of exosomes
And (3) detecting the grain size, distribution ratio, purity and morphology analysis of the exosomes obtained in the step (2.2) in the step (6.2) as shown in fig. 17-19, wherein the distribution ratio of gp17-YWHAQ-EGFP-Flag 293Evs in an exosome sample is about 13.1%, the grain size is 40-200 nm, the purity is more than 90%, and the exosomes are in a dish-shaped sinus membrane structure when observed by an electron microscope.
(2.4) gp17-YWHAQ-EGFP-Flag 293Evs targeting function validation
Mature liver organoid cells were incubated with gp17-YWHAQ-EGFP-Flag 293Evs and YWHAQ-EGFP-Flag 293Evs, respectively. And (3) detecting a flag label expressed by fusion of the scaffold, and imaging the surface binding effect of the exosomes and the liver organoids. The result shows that the targeted peptide exosome gp17-YWHAQ-EGFP-Flag 293Evs is tightly adsorbed on the surface of liver organoids to form a tight adsorption film. The non-targeting peptide exosomes diffuse into organoid cells, do not aggregate on the surface of hepatocytes, and are taken up by the organoids to different exosomes by statistics, which are significantly higher than exosomes containing the targeting peptide (as shown in fig. 20).
Example 7: targeting peptide exosomes enhances in vivo uptake of exosome-targeted organs
The targeted peptide exosomes gp17-YWHAQ-EGFP-Flag 293Evs prepared in example 6 were used as experimental groups, YWHAQ-EGFP-Flag 293Evs were used as control groups, and the animal model was wild type Balb/c mice, and exosomes were administered by tail vein injection.
Design of animal experiment
After single intravenous exosome-Nluc was observed, the samples were organized in Balb/c mice and the groupings are shown in table 1 below.
TABLE 1
The specific operation steps are as follows:
1) Weight of: weighing animals prior to their receipt and administration, respectively
2) Tissue material selection:
1h after administration, animal tissues, heart, liver, spleen, lung, brain and serum were taken. Group G1 is background group, G2 is experimental group, and G3 is control group. Weighing the total weight of the obtained tissues, uniformly taking about 20mg of each tissue, recording the accurate material weight, crushing and homogenizing by using grinding beads, centrifuging after crushing completely, and taking the supernatant for later use.
3) Tissue homogenate chemiluminescent intensity detection:
4) The Nanoluc enzyme activity in the homogenate is detected by adopting a Nano-Glo Luciferase Assay System kit, and the specific operation is shown in the specification of the kit.
5) The total amount of Nanoluc enzyme activity in each tissue was calculated and the total amount of exosomes taken up by each tissue was characterized.
The results of the calculations are shown below in figure 21, with targeted peptide exosomes increasing the distribution of exosomes in the liver compared to the control group.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (8)

  1. Use of a 14-3-3 protein theta isomer in the preparation of an extracellular vesicle scaffold protein, said 14-3-3 protein theta isomer having an amino acid sequence as set forth in SEQ ID No. 6, said preparation comprising over-expressing said 14-3-3 protein theta isomer in a cell producing said extracellular vesicle.
  2. 2. The use according to claim 1, wherein the extracellular vesicles are selected from one or more of exosomes, microvesicles, apoptotic bodies, tumor vesicles and nanovesicles.
  3. 3. The use according to claim 1, wherein the extracellular vesicles are extracellular vesicles localized to hepatocytes.
  4. 4. A method for preparing an extracellular vesicle, comprising overexpressing a polypeptide comprising a 14-3-3 protein theta isoform in a cell, allowing the 14-3-3 protein theta isoform to act as an extracellular vesicle scaffold protein, and isolating or producing the extracellular vesicle from the cell or a culture system comprising the cell;
    the amino acid sequence of the 14-3-3 protein theta isomer is shown in SEQ ID NO. 6;
    the cell comprises an expression system comprising:
    a first expression element encoding a scaffold protein, wherein the scaffold protein is the 14-3-3 protein theta isomer;
    and a second expression element encoding an active domain.
  5. 5. The method of claim 4, wherein the active domain is selected from the group consisting of a polypeptide and a polynucleotide.
  6. 6. The method of claim 4, wherein the scaffold protein is linked to the active domain directly or via a linker.
  7. 7. The method of claim 4, wherein the active domain is selected from any one or a combination of the following (i) - (iv):
    Antisense oligonucleotides, mRNA, siRNA, miRNA, shRNA, lncRNA, circRNA or any combination thereof;
    (ii) an antibody or antigen-binding fragment thereof, including a full-length antibody, scFv, (scFv) 2 、Fab、Fab'、F（ab'） 2 、F（ab1） 2 Fv, dAb and Fd fragments, bifunctional antibodies or any of themCombining;
    (iii) a nucleotide binding protein;
    (iv) fluorescent marker proteins and/or tag proteins.
  8. 8. The method of claim 7, wherein the nucleotide binding protein is selected from the group consisting of Ku protein, sm7 protein, MS2 coat protein, PP7 coat protein, and Com RNA binding protein.
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