CN118909084A - Extracellular vesicle scaffold protein and application thereof - Google Patents

Abstract

The present invention relates to the use of nerve growth factor receptor or its fragment as an extracellular vesicle scaffold protein. The present invention also relates to an extracellular vesicle scaffold protein and its use, the extracellular vesicle comprising a scaffold protein and a biologically active domain bound to the scaffold protein, the scaffold protein being a nerve growth factor receptor or its fragment. The present invention provides a new scaffold protein, which provides a new and more abundant connection basis for the drug loading or labeling of extracellular vesicles.

Description

Extracellular vesicle scaffold protein and its application

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to a scaffold protein for extracellular vesicles and an application thereof.

Background Art

Adeno-associated virus (AAV) is considered to be the safest and most promising drug delivery vector due to its extremely low immunogenicity. However, its small loading capacity (less than 4.7k) limits its application in some areas of gene therapy. Exosomes are nanoscale particles with low immunogenicity, making them suitable as a drug carrier. Moreover, the volume of exosomes is significantly larger than that of AAV. It has been reported in the literature that exosomes can accommodate 6k of nucleic acid, which has certain advantages over AAV. In natural exosomes, certain proteins are significantly enriched. These proteins can be used as scaffold proteins, and after a certain special protein is fused with the scaffold protein and expressed, this special protein will also be enriched, thereby achieving the loading of proteins and nucleic acids on exosomes. By delivering exosomes to recipient cells, the loaded proteins or nucleic acids can be exerted to achieve a therapeutic effect.

Some scaffold proteins commonly used in the prior art, such as CD63 and CD81, are unevenly distributed in exosomes, and only some exosomes contain such proteins. In drug delivery, the defects of the above scaffold proteins will affect the exosome delivery efficiency, thereby limiting the potential clinical application value of engineered exosomes. Therefore, it is necessary to find new scaffold proteins to improve the current status of exosome scaffold proteins.

Summary of the invention

In order to solve the above problems existing in the prior art, the present invention provides a scaffold protein for extracellular vesicles, which will not affect the growth of cells after overexpression, and the enrichment degree in extracellular vesicles is higher than that of the existing scaffold proteins used for treatment. At the same time, the distribution of the screened scaffold protein in extracellular vesicles is at least equivalent to that of commonly used scaffold proteins (such as CD63).

One aspect of the present invention provides the use of nerve growth factor receptor or a fragment thereof as an extracellular vesicle scaffold protein.

In some embodiments, the nerve growth factor receptor or its fragment is localized in an extracellular vesicle. In some embodiments, the nerve growth factor receptor or its fragment is localized on, within or outside the membrane of an extracellular vesicle. In some embodiments, the nerve growth factor receptor or its fragment is localized in the lumen of an extracellular vesicle.

In some embodiments, the nerve growth factor receptor 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 with SEQ ID NO:6.

In some embodiments, the nerve growth factor receptor has the amino acid sequence shown 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 with SEQ ID NO:1.

In some embodiments, the extracellular vesicles include one or more of exosomes, microvesicles, apoptotic bodies, tumor vesicles, and nanovesicles.

In some embodiments, the extracellular vesicle is a hepatocyte-localized extracellular vesicle.

Another aspect of the present invention provides an extracellular vesicle, which includes a scaffold protein, wherein the scaffold protein is a nerve growth factor receptor or a fragment thereof; and an active domain that binds to the scaffold protein.

In some embodiments, the nerve growth factor receptor 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 with SEQ ID NO:6.

In some embodiments, the nerve growth factor receptor has the amino acid sequence shown 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 with SEQ ID NO:1.

In some embodiments, the active domain comprises a polypeptide, a polynucleotide sequence, a compound, or any combination thereof.

In some embodiments, the active domain comprises an antisense oligonucleotide (ASO), siRNA, miRNA, shRNA, nucleic acid, or any combination thereof.

In some embodiments, the active domain comprises a peptide, a protein, an antibody or an antigen-binding fragment thereof, or any combination thereof. Preferably, the antigen-binding fragment of the antibody comprises scFv, (scFv) ₂ , Fab, Fab', F(ab') ₂ , F(ab1) ₂ , Fv, dAb and Fd fragments, bifunctional antibodies, antibody-related polypeptides, or any fragments thereof.

In some embodiments, the scaffold protein can be bound to the active domain directly or through a linker.

In some embodiments, the linker is a polypeptide linker and/or a non-polypeptide linker. In some embodiments, the linker can be a (chemical) covalent linker.

In some embodiments, the active domain comprises a nucleotide binding protein or a fragment thereof, wherein the scaffold protein forms a fusion protein with the nucleotide binding protein or a fragment thereof.

In some embodiments, the nucleotide binding protein or fragment thereof further binds to the polynucleotide sequence.

Preferably, the nucleotide binding protein is selected from Ku protein, Sm7 protein, MS2 coat protein, PP7 coat protein, Com RNA binding protein, aptamer ligand, or any combination, functional variant, fragment or domain thereof.

In some embodiments, the active domain can be displayed on the outer surface of the exosome or in the lumen of the exosome by using the scaffold protein nerve growth factor receptor or fragment thereof disclosed herein.

Another aspect of the present invention provides an expression system, comprising: a first expression element encoding a scaffold protein, wherein the scaffold protein is a nerve growth factor receptor or a fragment thereof; and a second expression element encoding an active domain.

In some embodiments, the nerve growth factor receptor 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 with SEQ ID NO:6.

In some embodiments, the nerve growth factor receptor has the amino acid sequence shown 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 with SEQ ID NO:1.

In some embodiments, the active domain comprises a polypeptide, a polynucleotide sequence, a compound, or any combination thereof.

In some embodiments, the active domain comprises an antisense oligonucleotide (ASO), siRNA, miRNA, shRNA, nucleic acid, or any combination thereof.

In some embodiments, the active domain comprises a peptide, a protein, an antibody or an antigen-binding fragment thereof, or any combination thereof. Preferably, the antigen-binding fragment of the antibody comprises scFv, (scFv) ₂ , Fab, Fab', F(ab') ₂ , F(ab1) ₂ , Fv, dAb and Fd fragments, bifunctional antibodies, antibody-related polypeptides, or any fragments thereof.

In some embodiments, the scaffold protein can be bound to the active domain directly or through a linker.

In some embodiments, the linker is a polypeptide linker and/or a non-polypeptide linker. In some embodiments, the linker can be a (chemical) covalent linker.

In some embodiments, the active domain comprises a nucleotide binding protein or a fragment thereof, wherein the scaffold protein forms a fusion protein with the nucleotide binding protein or the fragment thereof.

In some embodiments, the nucleotide binding protein or fragment thereof further binds to the polynucleotide sequence.

Preferably, the nucleotide binding protein is selected from Ku protein, Sm7 protein, MS2 coat protein, PP7 coat protein, Com RNA binding protein, aptamer ligand, or any combination, functional variant, fragment or domain thereof.

In some embodiments, the expression system can be selected from a prokaryotic expression system, a eukaryotic expression system or a viral expression system. In some embodiments, the expression system can include a prokaryotic vector, a eukaryotic vector or a viral vector, etc.

Another aspect of the present invention provides a host cell, wherein the host cell comprises the above expression system.

In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the prokaryotic cell can be selected from Escherichia coli or Bacillus subtilis, such as Escherichia coli BL21, T7E, C41, Arctic, etc. In some embodiments, the eukaryotic cell can be selected from yeast cells, insect cells, plant cells, mammalian cells, etc., such as yeast cells, CHO cells, 293 cells, Vero cells, NSO cells, etc.

Another aspect of the present invention provides a pharmaceutical composition, comprising the extracellular vesicles described in the second aspect and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is in the form of tablets, powders, granules, pills, injections, suspensions, powders, emulsions, aerosols, gels, eye drops, sustained release agents or sustained release implants. In some embodiments, the pharmaceutical composition can be formulated into an injectable formulation. In some embodiments, the formulation is suitable for intravitreal injection, subcutaneous, intradermal, intramuscular, intravenous, intrathecal or intraspinal administration.

Another aspect of the present invention provides use of the extracellular vesicles of the present invention in preparing a medicament for preventing or treating a disease.

In some embodiments, the disease comprises cancer, an inflammatory disorder, a neurodegenerative disorder, a central nervous system disease, or a metabolic disease.

In some embodiments, the extracellular vesicles are administered intravenously, intraperitoneally, nasally, orally, intramuscularly, subcutaneously, parenterally, or intratumorally.

In some embodiments, the extracellular vesicles can be used for drug loading and/or marker loading.

The invention provides a new scaffold protein, which provides a new connection basis with higher abundance for drug loading or labeling of extracellular vesicles.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the TEM results of exosomes overexpressing different scaffold proteins.

FIG2 shows the markers (Western Blot) results of exosomes overexpressing different scaffold proteins.

Figure 3 shows the HPLC purity characterization results after purification of different exosomes, where a is 293Evs, b is NGFR-Evs, and c is CD63-293Evs.

Figure 4 shows the HPLC purity characterization results after purification of different exosomes, where a is MFGE8-Evs, b is PTGFRN-Evs, and c is Basp-1-293Evs.

Figure 5 shows the characterization results of the scaffold distribution ratios of different exosomes.

Figure 6 shows the results of characterization of scaffold abundance of different exosomes.

FIG. 7 shows the purity (a), morphology (b), particle size (c) and EGFP protein loading (d) characterization results of exosomes overexpressing truncated-NGFR scaffold protein.

Figure 8 shows the characterization results of the purity (a), particle size (b) and morphology (c) of exosomes NGFR-MCP-MS2-FIX-Fc293Evs after loading mRNA using the RNA-binding protein MCP.

Figure 9 shows the characterization results of the purity (a), particle size (b) and morphology (c) of exosome NGFR-L7Ae-C/D box-FIX-Fc293Evs after loading mRNA using the RNA binding protein L7Ae.

FIG. 10 shows the purity (a), particle size (b) and morphology (c) characterization results of exosome NGFR-FIX-Fc 293Evs after mRNA was not loaded using RNA binding protein.

FIG. 11 shows the purity (a), particle size (b) and morphology (c) characterization results of exosome NGFR 293Evs that are not loaded with mRNA but only overexpress the NGFR scaffold.

FIG. 12 shows the mRNA loading characterization results of NGFR fused to different RNA binding proteins.

Figure 13 shows the characterization results of the purity (a), particle size (b), morphology (c) and antibody positivity (d) of NGFR targeting antibody-displaying exosomes CD19scfv-NGFR-EGFP 293Evs.

FIG. 14 shows the characterization results of the purity (a), particle size (b), morphology (c) and antibody positivity (d) of exosomes NGFR 293Evs that do not display targeting antibodies.

FIG. 15 shows the characterization results of the purity (a), particle size (b), morphology (c) and antibody positivity (d) of exosomes 293Evs from expi 293F cells that do not overexpress NGFR scaffolds.

FIG. 16 shows the ELISA column results of the binding of CD19scfv on the surface of exosomes to CD19 antigen.

Figure 17 shows the characterization results of the exosome purity (a), particle size (b), targeting peptide positive ratio (c) and morphology (d) of NGFR targeting peptide displaying exosomes gp17-NGFR-EGFP-Flag 293Evs.

FIG. 18 shows the characterization results of the exosome purity (a), particle size (b), targeting peptide positive ratio (c) and morphology (d) of NGFR exosomes NGFR-EGFP-Flag 293Evs that do not display the targeting peptide.

FIG. 19 shows the characterization results of the exosome purity (a), particle size (b), targeting peptide positive ratio (c) and morphology (d) of exosomes 293Evs of expi 293F cells that do not overexpress NGFR scaffolds.

FIG20 shows the results of NGFR targeting peptide exosome targeting functional organoid detection.

FIG. 21 shows the phagocytic distribution results of NGFR targeting peptide exosomes in liver organoids.

FIG. 22 shows the effect of exosomes containing NGFR targeting peptides on the in vivo distribution of exosomes in mice.

DETAILED DESCRIPTION

Exosomes are extracellular vesicles with a diameter of 30 to 200 nm secreted by cells. They are rich in RNA (miRNA, lncRNA, circRNA, etc.), DNA, proteins and lipids, and participate in intercellular molecular transmission. Exosomes are a highly heterogeneous group, including size heterogeneity, content heterogeneity, functional heterogeneity, and source heterogeneity. Therefore, the types and quantities of molecules rich in exosomes produced by different cells are different, and the types and quantities of molecules rich in exosomes separated by different exosome purification methods are also different. Exosomes are rich in various molecules. When taken up by cells, they will have different degrees of impact on recipient cells. Natural exosomes from different cells will have some functions of the source cells. By modifying the cells, the function of exosomes is affected and used for treatment or drug delivery.

Nerve growth factor receptor (NGFR) belongs to the tumor necrosis factor transmembrane receptor superfamily and is a transmembrane low-affinity receptor of the neurotrophin family. NGFR is known to bind to nerve growth factor (NGF) and other neurotrophic factors, stimulate nerve growth and regulate the differentiation of sympathetic neurons and some sensory neurons. The present disclosure unexpectedly found that NGFR can be enriched in exosomes and can be used as an exosome scaffold protein to carry exogenous proteins or nucleic acids to achieve the desired effect.

In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

The terms "extracellular vesicle", "exovesicle" or "EV" as used herein refer to cell-derived vesicles comprising a membrane encapsulating an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, microvesicles, apoptotic bodies, tumor vesicles, nanovesicles, etc.), whose diameter is smaller than the diameter of the cell from which it is derived. In some aspects, the diameter of the extracellular vesicle is in the range of 20nm to 1000nm, and may be contained in the internal space (i.e., cavity), displayed on the outer surface of the extracellular vesicle and/or across the membrane. Various macromolecular payloads. In some aspects, the payload may include nucleic acids, proteins, carbohydrates, lipids, small molecules and/or combinations thereof. In some aspects, the extracellular medium comprises a scaffold portion. As an example and not limitation, extracellular vesicles include apoptotic bodies, cell fragments, cell-derived vesicles obtained by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles of vesicles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). The extracellular vesicles can 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 expressing one or more transgenic products.

As used herein, the term "exosome" refers to an extracellular vesicle with a diameter between 20 and 300 nm (e.g., between 40 and 200 nm). Exosomes comprise a membrane enclosing an internal space, and in some aspects, can be produced by cells by direct plasma membrane budding or by fusion of late endosomes or multivesicular bodies with the plasma membrane. In certain aspects, exosomes comprise a scaffold portion. As described below, exosomes can be derived from producer cells and isolated from producer cells based on their size, density, biochemical parameters, or a combination thereof. In some aspects, the EVs (e.g., exosomes) of the present disclosure are produced by cells expressing one or more transgene products.

The term "nanovesicle" as used herein refers to an extracellular vesicle having a diameter between 20-250nm (e.g., between 30-150nm), and is produced by a cell (e.g., a production cell) by direct or indirect manipulation, so that the cell will not produce nanovesicles without manipulation. Suitable manipulations of cells for the production of nanovesicles include, but are not limited to, continuous extrusion, treatment with an alkaline solution, ultrasonic treatment, or a combination thereof. In some aspects, the nanovesicle population described herein is substantially free of vesicles derived from cells by direct budding from the plasma membrane or late endosome fusion with the plasma membrane. In some aspects, the nanovesicle comprises a scaffold portion, such as scaffold X and/or scaffold Y. Nanovesicles, once derived from production cells, can be separated from production cells based on their size, density, biochemical parameters, or a combination thereof.

The term "fusion protein" as used herein refers to two or more proteins or fragments thereof co-linearly linked through their respective peptide backbones using genetic expression of polynucleotides encoding the proteins or protein synthesis methods.

The term "antibody" as used herein encompasses immunoglobulins (whether produced naturally or partially or completely synthetically) and fragments thereof. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain. "Antibody" also includes polypeptides comprising 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 complete antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, fragments thereof, and also includes single chain antibodies, humanized antibodies, murine antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotypic 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 as long as they exhibit the desired biological activity or function.

As used herein, the terms "active domain" or "biologically active domain" are used interchangeably and refer to any molecule that can be linked to a scaffold protein via an anchoring moiety, wherein the molecule can have a therapeutic or prophylactic effect in a subject in need thereof, or can be used for diagnostic purposes. Thus, for example, the term biologically active domain includes proteins (e.g., antibodies, proteins, polypeptides, and derivatives, fragments, and variants thereof), polynucleotide sequences, or chemical compounds, etc. In some embodiments, the biologically active domain includes antisense oligonucleotides (ASOs), siRNAs, miRNAs, shRNAs, or nucleic acids. In some embodiments, the antigen-binding fragments of antibodies include scFv, (scFv) ₂ , Fab, Fab', F(ab') ₂ , F(ab1) ₂ , Fv, dAb and Fd fragments, bifunctional antibodies, or antibody-related polypeptides.

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 triple-stranded, double-stranded, and single-stranded deoxyribonucleic acids ("DNA"), and triple-stranded, double-stranded, and single-stranded ribonucleic acids ("RNA"). It also includes modified (e.g., by alkylation and/or by capping) and unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose); polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced or unspliced; any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base; and other polymers containing an orthonucleotide backbone, such as polyamides (e.g., peptide nucleic acids "PNA") 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. 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 with a labeling component. Also included in the definition are, for example, polypeptides containing 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 term "polypeptide" as used herein refers to proteins, polypeptides and peptides of any size, structure or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologues, orthologues, lateral homologues, fragments of the aforementioned and other equivalents, variants and analogs. Polypeptides can be single polypeptides or can be multimolecular complexes, such as dimers, trimers or tetramers. They can also contain single-chain or multi-chain polypeptides. Most commonly, disulfide bonds are present in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some aspects, a "peptide" can 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.

As used herein, the term "connector" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some embodiments, two or more connection sons can be connected in series. When multiple connection sons are present, each connection son can be the same or different. In general, the connection son provides flexibility or prevents/improves steric hindrance. The connection son is usually not cut; however, in some aspects, such cutting may be required. Therefore, in some embodiments, the connection son may include one or more protease cleavable sites, which may be located within the connection son sequence or at the connection son flank at either end of the connection son sequence.

In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker can include 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 embodiments, the linker includes one or more amino acids. In some embodiments, the linker includes a Gly-Ser (GS) linker. In some embodiments, the GS linker includes (G4S)n, wherein n is an integer between 1 and 10. In some embodiments, the GS linker includes (G3S)n, wherein n is an integer between 1 and 10.

In some embodiments, the peptide linker is synthetic, i.e., non-naturally occurring. In one aspect, the peptide linker comprises a peptide (or polypeptide) (e.g., a naturally or non-naturally occurring peptide) comprising an amino acid sequence that connects or genetically fuses a first linear amino acid sequence to a second linear amino acid sequence, wherein the first linear amino acid sequence is not naturally connected or genetically fused to the second linear amino acid sequence in nature. For example, in one aspect, the peptide linker may comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation such as an addition, substitution, or deletion).

The linker may be easily cleaved (a "cleavable linker"), thereby facilitating the release of the exogenous biologically active portion. In some embodiments, the linker is a "reduction-sensitive linker". In some embodiments, the reduction-sensitive linker contains a disulfide bond. In some embodiments, the linker is an "acid-labile linker". In some embodiments, the acid-labile linker contains a hydrazone. Suitable acid-labile linkers also include, for example, cis-aconitic acid linkers, hydrazide linkers, thiocarbamoyl linkers, or any combination thereof. In some embodiments, the ASO is associated with an EV (e.g., an exosome) with the aid of a linker. In some embodiments, the linker includes acrylic acid phosphoramidite (e.g., ACRYDITE™), adenylation, azide (NHS Ester), digoxigenin (NHS Ester), cholesterol-TEG, I-LINKER™, an amino modifier (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, or Uni-Link™ amino modifier), alkyne, 5' hexynyl, 5-octadiynyl dU, biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, or desthiobiotin), thiol modification (thiol modifier C3 S-S, dithiol, or thiol modifier C6 S-S), or any combination thereof.

In some embodiments, the linker comprises a terpene, such as nerolidol, farnesol, limonene, linalool, geraniol, carvone, fenchone, or menthol; a lipid, such as palmitic acid or myristic acid; cholesterol; oleyl; retinyl; a cholesterol residue; cholic acid; adamantaneacetic acid; 1-pyrenebutyric acid; dihydrotestosterone; 1,3-bis-O (hexadecyl) glycerol; geranyloxyhexyl; hexadecylglycerol; borneol; 1,3-propanediol; heptadecyl; O3-(oleoyl) lithocholic acid; O3-(oleoyl) cholic acid; dimethoxytrityl; phenoxazine, a 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 the membrane of the EV (e.g., exosome). In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker includes tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), succinimide or any combination thereof. In some embodiments, the linker includes a spacer unit that connects a bioactive molecule to the linker. In some embodiments, one or more linkers include smaller units (e.g., HEG, TEG, glycerol, C2 to C12 alkyl, etc.) connected together. In one aspect, the linkage is an ester linkage (e.g., phosphodiester or thiophosphate) or other linkage. In some embodiments, the linker includes polyethylene glycol (PEG), characterized in that the formula R3-(O-CH2-CH2)n- or R3-(0-CH2-CH2)n-O-, wherein R3 is hydrogen, methyl or ethyl, and the value of n is 2 to 200. In some embodiments, the linker includes a spacer, wherein the spacer is PEG. In some embodiments, the PEG linker is an oligoethylene glycol, such as a diethylene glycol, triethylene glycol, tetraethylene glycol (TEG), pentaethylene glycol, or hexaethylene glycol (HEG) linker.

As used herein, the term "prevent" or "prevent" refers to partially or completely delaying the onset of a disease, disorder, and/or condition; partially or completely delaying the onset of one or more symptoms, features, or clinical manifestations of a specific disease, disorder, and/or condition; partially or completely delaying the onset of one or more symptoms, features, or manifestations of a specific disease, disorder, and/or condition; partially or completely delaying the progression from a specific disease, disorder, and/or condition; and/or reducing the risk of developing pathology associated with a disease, disorder, and/or condition.

As used herein, the term "treat" refers to, for example, a reduction in the severity of a disease or disorder; a shortening of the duration of a disease course; an improvement or elimination of one or more symptoms associated with a disease or disorder; providing a beneficial effect to a subject suffering from a disease or disorder, but not necessarily curing the disease or disorder. The term also includes the prevention or prophylaxis of a disease or disorder or its symptoms.

As used herein, the term "replacement" or "substitution" of an amino acid may refer to a conservative amino acid substitution, wherein an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues 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 substituted with another amino acid from the same side chain family, the substitution is considered conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in the order and/or composition of 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 the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, gaps present in the target sequence are not counted. Likewise, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The determination of the sequence identity percentage between the comparison of the sequence and the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical preparation other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The scaffold protein provided by the present invention can be used as a scaffold protein on exosomes, and can be used to connect drugs or markers to achieve drug loading function or tracing research. In certain embodiments of the present invention, the scaffold protein can also be modified, such as truncation, mutation, insertion of exogenous sequences or modification. In some embodiments, the N-terminus of the scaffold protein is fused with a short peptide chain, and the short peptide chain can express a targeting peptide, a recombinant peptide, a therapeutic peptide or a linker. In other embodiments, the C-terminus of the scaffold protein is fused with a short peptide chain, and the short peptide chain can express a therapeutic peptide, a recombinant peptide or a linker. In some embodiments, the C-terminus of the scaffold protein is fused with an RNA binding protein, and the RNA binding protein can bind to an RNA containing a special domain, and the RNA containing a special domain contains a functional region with a medicinal effect and a connection region for connecting to the RNA binding protein; the RNA used for treatment can be mRNA, miRNA, LncRNA, circRNA or siRNA

Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

Example 1. Expression vector construction

The NGFR protein (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), and MFGE8 (SEQ ID NO: 5) were used to construct expression plasmids pcDNA3.1 (+) -scaffold protein-EGFP-Nanoluc, respectively.

Example 2. Preparation and verification of exosomes

Exosomes expressing EGFP-Nanoluc fused with scaffold NGFR, BASP-1, PTGFRN, CD63 and MFGE8 expression proteins as well as natural Expi 293F exosomes (named NGFR-293Evs, BASP-1-293Evs, PTGFRN-293Evs, CD63-293Evs, MFGE8-293Evs and 293Evs) were prepared respectively.

2.1 293s cell passaging

The cell recovery density of Expi 293F cells is 3-10×10 ⁵ cells/ml, and the cell density is controlled at 0.5-5×10 ⁶ cells/ml during passaging.

2.2 Cell transfection

Before transfection, the cell number of the passaged cells in step 2.1 was adjusted to 1-3×10 ⁶ cells/ml, the plasmid concentration was 1-1.5 μg/ml, and the concentration ratio of polyethyleneimine (PEI) to plasmid was controlled to be (1:1)-(4:1). The four plasmids obtained in step (1.2) were transfected into 293s cells according to the above conditions to overexpress proteins NGFR, BASP-1, PTGFRN, CD63 and MFGE8 in the cells. The cells were cultured at 37°C and 5% CO ₂ for 48-72 hours, and the cell culture supernatant was collected for downstream purification.

2.3 Exosome purification

The cell culture supernatant collected in step 2.2 was used for ultrafiltration, density gradient centrifugation, and molecular sieve filtration to obtain exosome samples that can be used for nanoflow cytometry analysis.

2.4 Exosome quality inspection

The purified exosomes were characterized, and the morphological (TEM) characterization results are shown in Figure 1. The purified product obtained had a typical exosome disc-shaped sinus membrane layer structure.

In addition, the expression of exosome markers CD81 and TSG101, Golgi marker GM130 and endoplasmic reticulum marker Calexin from each group of cells was detected by Western Blot, and the results are shown in Figure 2. The purity (HPLC) characterization results are shown in Figures 3 and 4. It can be seen from Figures 2 to 4 that the exosomes obtained from each group of cells highly expressed CD81 and TSG101, and the expression of GM130 and Calexin was not detected, further indicating that the exosomes obtained were exosomes with high purity.

Example 3. Distribution and abundance characterization of exosomes engineered with new scaffold proteins on exosomes

3.1 Comparison of exosome scaffold distribution characterization

Nano-flow was used to detect the proportion of FITC-positive particles in 20 μL exosomes of different scaffold proteins and the negative control group, as shown in Figure 5. Figure 5 shows that the NGFR-positive exosome subpopulation accounts for more than 50% of the entire exosome distribution, which is higher than the currently disclosed exosome universal scaffold CD63.

3.2 Comparison of exosome scaffold abundance characterization

use The reagents were used to detect the Nanoluc enzyme activity in the exosomes with the same number of particles of different scaffold proteins and the negative control group, and then the abundance of the fusion expression scaffolds in the exosomes with the same number of particles was characterized, as shown in Figure 6. Figure 6 shows that the abundance of the NGFR scaffold is greater than that of the CD63 scaffold.

Example 4: A new scaffold protein truncated protein that achieves exosome localization and loading of foreign substances

In order to determine the shortest sequence of NGFR to achieve exogenous substance loading, this example is divided according to the protein structure of NGFR, based on the internal and external domains of the transmembrane segment, and gradually reduces the domains outside and inside the membrane. Finally, the amino acids 200-338 are determined and named as truncated-NGFR (SEQ ID NO: 6), which is the shortest sequence to achieve exosome localization loading.

The following is the verification of its distribution and abundance in exosomes.

4.1 Expression vector construction and preparation

(1.1) Vector construction

The C-terminus of truncated-NGFR was fused with EGFP-Nanoluc to construct and verify the expression vector pcDNA3.1-truncated-NGFR-EGFP-Nanoluc.

(1.2) Plasmid preparation

Plasmid preparation was the same as previously commissioned.

(1.3) Exosome preparation

The plasmid prepared in (1.2) was transfected into Expi 293 cells, and the preparation, purification and quality control process were the same as in Example 2. It was named truncated-NGFR-293Evs.

4.2Verification of truncated-NGFR-293Evs loading EGFP into exosomes

The high-purity exosomes prepared in (1.3) were characterized. Figure 7 shows that the purity of the exosomes is close to 100%, the morphology is a disc-shaped sinus membrane structure, and the particle size distribution range is within the normal range of exosomes. The exosome EGFP positivity rate is about 19%, indicating that truncated-NGFR can load EGFP into exosomes, retaining the localization and loading functions of exosomes.

Example 5: NGFR as a scaffold protein fused to RNA binding protein for loading mRNA

In this embodiment, the RNA binding proteins selected are the RNA binding protein MCP protein of MS2 bacteriophage (SEQ ID NO: 7) and L7Ae of archaea (SEQ ID NO: 8), whose recognition sequences are MS2 (SEQ ID NO: 9) and C/D box sequence (SEQID NO: 10), respectively, and the selected mRNA is the mRNA encoding the fusion protein of protein coagulation factor 9 and Fc.

5.1 Expression vector construction and preparation (1.1) Vector construction

1) RNA binding proteins MCP and L7Ae were fused to the C-terminus of NGFR, respectively, and scaffold expression plasmids pcDNA3.1(+)-scaffold protein-MCP/L7Ae (RNA binding protein) were constructed. NGFR and RNA binding protein were fused and expressed to make NGFR a scaffold capable of loading mRNA. pcDNA3.1(+)-scaffold protein was used as a control.

2) The RNA binding protein recognition sequence (MCP recognizes MS2, L7Ae recognizes C/D box) is positioned at the 3' end of the loaded mRNA, after the stop codon and before polyA, to construct the mRNA transcription plasmid pcDNA3.1-FIX-Fc-MS2/C/Dbox (binding protein recognition sequence), pcDNA3.1-FIX-Fc is the corresponding plasmid to the control.

(1.2) Plasmid preparation

Plasmid preparation was the same as previously commissioned.

5.2 Exosome preparation

The scaffold expression plasmid (pcDNA3.1 (+) -scaffold protein -MCP / L7Ae) prepared in 5.1 was co-transfected with the mRNA transcription plasmid (pcDNA3.1-FIX-Fc-MS2 / C / Dbox) to prepare exosomes loaded with mRNA. The preparation steps were the same as in Example 2, and 4 groups of exosomes were prepared, named NGFR-MCP-MS2-FICX-Fc 293Evs, NGFR-L7Ae-C / Dbox-FIX-Fc 293Evs, NGFR-FIX-Fc 293Evs and NGFR-293Evs, and the results of analysis of their morphology, particle size and purity are shown in Figures 8-11. Figures 8-11 show that the purity of the exosomes is close to 100%, the morphology is the exosome disc-shaped sinus membrane layer disc-shaped sinus membrane layer disc-shaped sinus layer structure, and the particle size distribution range is the normal range of exosomes.

5.3 Characterization of exosome-loaded target (FIX 9) mRNA

Calculation of FIX 9 mRNA in exosome samples:

1) Take 200 μl of exosomes to extract total RNA.

2) Perform RT-qPCR on RNA to detect the copy number of FIX mRNA in RNA.

3) Detect the exosome particle number concentration corresponding to each sample.

4) Calculate the average mRNA copy number in a single exosome.

As shown in Figure 12, the exosomes NGFR-MCP-MS2-FICX-Fc 293Evs and NGFR-L7Ae-C/D box-FIX-Fc 293Evs that fused and expressed the RNA-binding protein scaffold can increase the mRNA loading amount compared with the NGFR-FIX-Fc 293Evs exosomes. It can be seen that after the fusion of the scaffold protein and the RNA-binding protein, more mRNA can be packaged into the exosomes, thereby realizing the scaffold protein mRNA loading empowerment.

Example 6: Exosome targeting function

6.1 Exosome surface targeted antibody display (1.1) Construction of scaffold fusion expression antibody plasmid

Using NGFR as an antibody display scaffold, an expression vector was constructed to display antibodies on the surface of exosomes distributed by the chimeric scaffold to achieve the exosome targeting function. The displayed antibody is the human CD19 corresponding clone number FMC63 antibody. The plasmid name is pcDNA3.1-CD19scfv-NGFR-EGFP.

(1.2) Exosome preparation

Exosomes expressing CD19scfv (named CD19scfv-NGFR-EGFP 293Evs), exosomes overexpressing only scaffold protein (named NGFR-EGFP 293Evs), and 293F cell exosomes (named 293Evs) were prepared respectively.

The process of 293s cell passaging, cell transfection and exosome purification was the same as in Example 1.

(1.3) Exosome quality inspection

The morphology, particle size and purity of the sample obtained in step (1.2) were analyzed and tested. As shown in Figures 13-15 below, the distribution ratio of CD19scfv-NGFR-EGFP 293Evs in the prepared sample was about 60%, the particle size was between 40nm-200nm, the purity was greater than 90%, and the exosome morphology was observed by electron microscopy to be an exosome disc-shaped sinus membrane layer disc-shaped sinus membrane layer structure.

(1.4) Functional verification of CD19scfv-NGFR-EGFP 293Evs targeting CD19 antigen

1) CD19 antigen was coated on the solid phase carrier Elisa plate at 4°C overnight;

2) After washing with PBST buffer, the plate was blocked with 2% BSA at room temperature for 2 h;

3) After washing the plate, incubate an equal amount of the exosome sample to be tested at room temperature for 1 h;

4) After washing, incubate with the subsequent exosome surface marker CD9 antibody, followed by HRP-labeled secondary antibody;

5) After the substrate develops color, the reaction is terminated and the sample absorbance is detected by OD450 to evaluate whether the detected exosome sample is CD19 antigen-bound exosome.

The results in Figure 16 show that CD19scfv on the surface of exosomes can specifically bind to the CD19 antigen, thereby realizing the exosome antigen targeting function.

6.2 Targeting peptide display on exosome surface (2.1) Construction of scaffold fusion expression targeting peptide plasmid

The exosome distribution protein NGFR was used as a targeting peptide display scaffold, and the displayed targeting peptide was a short peptide targeting hepatocytes and derived from bacteriophage P17 protein, so as to achieve exosome targeting hepatocytes. The plasmid was named pcDNA3.1-gp17-NGFR-EGFP.

(2.2) Preparation of exosomes

Exosomes expressing gp17 targeting peptide (named gp17-NGFR-EGFP-Flag 293Evs), exosomes without targeting peptide (named NGFR-EGFP-Flag 293Evs) and 293 original exosomes (named 293Evs) were prepared respectively.

The process of 293s cell passaging, cell transfection and exosome purification was the same as in Example 1.

(2.3) Quality control of exosomes

The particle size, distribution ratio, purity and morphological analysis test data of the exosomes obtained in step (2.2) are shown in Figures 17-19. The distribution ratio of gp17-NGFR-8 293Evs in the exosome sample is approximately 30%, the particle size is between 40nm and 200nm, the purity is greater than 90%, and the exosome morphology observed by electron microscopy is an exosome disc-shaped sinus membrane layer structure.

(2.4) Verification of gp17-NGFR-EGFP-Flag 293Evs targeting function

Gp17-NGFR-EGFP-Flag 293Evs and NGFR-EGFP-Flag 293Evs were incubated with mature liver organoid cells respectively. The surface binding effect of exosomes and liver organoids was imaged by detecting the flag tag expressed by the scaffold fusion. As shown in Figures 20-21 below, the targeted peptide exosomes gp17-NGFR-8-EGFP-Flag 293Evs were tightly adsorbed on the surface of liver organoids to form a tight adsorption film. However, the non-targeted peptide exosomes diffused in the organoid cells and did not aggregate on the surface of liver cells.

Example 7: Targeted peptide exosomes improve in vivo uptake of exosomes into targeted organs

The targeting peptide exosomes gp17-NGFR-EGFP-Flag 293Evs prepared in Example 6 were used as the experimental group, and NGFR-EGFP-Flag 293Evs were used as the control group. The animal model was wild-type Balb/c mice, and the exosomes were administered by tail vein injection.

Animal experiment design

The tissue distribution of the test article in Balb/c mice after a single intravenous injection of exosome-Nluc was observed, and the groups were shown in Table 1 below.

Table 1

The specific steps are as follows:

1) Body weight: Weigh the animals at the time of receipt and before administration.

2) Tissue sampling:

One hour after administration, animal tissues, heart, liver, spleen, lung, brain and serum were collected. Group G1 was the background group, G2 was the experimental group, and G3 was the control group. The total weight of the tissues was weighed, and about 20 mg of each tissue was evenly collected, and the accurate weight of the collected tissue was recorded. The grinding beads were crushed and homogenized, and the supernatant was taken for later use after complete crushing.

3) Chemiluminescence intensity detection of tissue homogenate:

4) Adoption Luciferase Assay System kit is used to detect Nanoluc enzyme activity in the homogenate. For specific operations, please refer to the kit instructions.

5) Calculate the total amount of Nanoluc enzyme activity in each tissue to characterize the total amount of exosomes taken up by each tissue.

The calculation results are shown in Figure 22 below. Compared with the control group, the targeted peptide exosomes increased the distribution of exosomes in the liver.

The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims (14)

1. Use of a nerve growth factor receptor or fragment thereof as an extracellular vesicle scaffold protein.

2. The use according to claim 1, wherein said nerve growth factor receptor has at least the amino acid sequence shown in SEQ ID No. 6 or an amino acid sequence having at least 85% sequence identity with SEQ ID No. 6.

3. The use according to claim 1, wherein the nerve growth factor receptor has the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence having at least 85% sequence identity to SEQ ID No. 1.

4. The use of claim 1, wherein the extracellular vesicles comprise 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 nerve growth factor receptor or fragment thereof is localized to an extracellular vesicle.

5. An extracellular vesicle, wherein the extracellular vesicle comprises:

a scaffold protein, which is a nerve growth factor receptor or fragment thereof; and

An active domain.

6. The extracellular vesicle according to claim 5, wherein said nerve growth factor receptor has at least the amino acid sequence shown in SEQ ID No.6 or an amino acid sequence having at least 85% sequence identity with SEQ ID No. 6.

7. The extracellular vesicle according to claim 5, wherein said nerve growth factor receptor has an amino acid sequence shown in SEQ ID No.1 or an amino acid sequence having at least 85% sequence identity with SEQ ID No. 1.

8. The extracellular vesicle according to claim 5, wherein the active domain comprises a polypeptide, a polynucleotide sequence, a compound, or any combination thereof,

Preferably, the active domain comprises an antisense oligonucleotide (ASO), siRNA, miRNA, shRNA, a nucleic acid, or any combination thereof,

Preferably, the active domain comprises a peptide, a protein, an antibody or antigen binding fragment thereof or any combination thereof,

Preferably, the antigen-binding fragment of the antibody comprises 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; and/or

The scaffold protein binds to the active domain directly or through a linker.

9. The extracellular vesicle according to claim 8, wherein the active domain comprises a nucleotide binding protein or fragment thereof, wherein the scaffold protein forms a fusion protein with the nucleotide binding protein or fragment thereof,

Preferably, the nucleotide binding protein or fragment thereof further binds to the polynucleotide sequence,

Preferably, the nucleotide binding protein is selected from Ku protein, sm7 protein, MS2 coat protein, PP7 coat protein, com RNA binding protein, aptamer ligand, or any combination, functional variant, fragment or domain thereof.

10. An expression system, the expression system comprising:

a first expression element encoding a scaffold protein, wherein the scaffold protein is a nerve growth factor receptor or fragment thereof; and

A second expression element encoding an active domain.

11. A host cell comprising the expression system of claim 10.

12. A pharmaceutical composition comprising the extracellular vesicles of any one of claims 5 to 9 and a pharmaceutically acceptable carrier.

13. Use of an extracellular vesicle according to any one of claims 5 to 9 in the manufacture of a medicament for the prevention or treatment of a disease.

14. The use according to claim 13, wherein the disease comprises cancer, an inflammatory disorder, a neurodegenerative disorder, a central nervous disease or a metabolic disease, and/or

The extracellular vesicles are administered intravenously, intraperitoneally, nasally, orally, intramuscularly, subcutaneously, parenterally or intratumorally.