CN116769718A - An engineered extracellular vesicle and its use - Google Patents

Abstract

The present invention provides methods for loading biologically active substances in extracellular vesicles, especially exosomes. The method includes generating lumen-engineered exosomes that include one or more higher concentrations of exosomal proteins, modifications or fragments of exosomal proteins, or fusion proteins of exosomal proteins with therapeutic or cargo proteins. The manufacturing method includes generating engineered exosomes that contain one of a protein, a modification or fragment of the protein, or a fusion protein of the protein and a payload at a higher concentration than that observed in wild-type exosomes. Kind or variety.

Description

An engineered extracellular vesicle and its use

Technical field

The invention belongs to the field of genetic engineering and provides an extracellular vesicle rich in scaffold proteins, especially exosomes, which can be used to prevent or treat cancer and other diseases.

Background technique

Exosomes are important mediators of intercellular communication. They are also important biomarkers in the diagnosis and prognosis of many diseases, such as cancer. In the pharmaceutical field, exosomes are often used as drug delivery vehicles. As a new treatment modality, exosomes offer many advantages over conventional drug delivery methods in many therapeutic areas.

The main characteristic of exosomes is the ability to contain bioactive payloads within their internal space or lumen. Exosomes are known to contain endogenous payloads, including mRNA, miRNA, DNA, proteins, carbohydrates, and lipids, but the ability to specifically load desired therapeutic payloads is currently limited. Exosomes can be loaded by overexpressing the desired therapeutic payload in producer cells, but the efficiency of this loading is often limited due to the random localization of the payload to the cell's exosome processing centers. Furthermore, purified exosomes such as siRNA can be loaded ex vivo, for example by electroporation. These methods may be inefficient or limited to small payloads. Therefore, the generation of efficient and clear loaded exosomes can better realize therapeutic uses and other applications based on exosome technology.

Contents of the invention

Extracellular vesicles (EVs), such as exosomes, small vesicles, and large vesicles, contain a variety of conventional endogenous proteins. Taking exosomes as an example, these proteins can regulate the interaction between the exosome membrane and the receptor cell. Fusion, exosome membrane exchange and fusion, etc., the inventor screened and identified a group of proteins on the exosome membrane surface, overexpressed the required proteins in the production cells, and enriched these proteins as scaffold proteins in the exosomes. In the body, especially on the exosome membrane, it has a significantly higher concentration compared with endogenous proteins.

The screened and identified scaffold proteins include: ENPP1, Rab7a, STX7, STX4, EPCAM, CXADR, TRFC, TRFC-81, CD55, IST1, VTA1, SNAP23, AT1A1, PDL1, VAMP2, and Sytenin.

Among the above-mentioned scaffold proteins, ENPP1, Rab7a, EPCAM, STX7, and CXADR have been proven to have preferred effects. In particular, ENPP1 has good effects in all aspects, with excellent loading levels, short cell phagocytosis time, and phagocytosis efficiency. .

ENPP1 (SEQ ID NO:1) is a protein containing 925 amino acids, which contains N-terminal cytoplasmic domain (CD), transmembrane domain (TM), growth hormone B-like domain 1 (SMB1), growth hormone B like domain 2 (SMB2), phosphodiesterase catalytic domain, nuclease-like domain.

Furthermore, ENPP1 truncations were shown to be sufficient to direct efficient loading of fluorescent protein cargo molecules to a higher extent than ENPP1 full-length protein loading.

Truncation was performed based on the structural domain of the ENPP1 protein. Specifically, truncation 596 (SEQ ID NO: 5) contained CD, TM, SMB1, SMB2, and phosphodiesterase catalytic domain. Truncated 190 (SEQ ID NO:4) contains CD, TM, SMB1 and SMB2 domains. Truncated 144 (SEQ ID NO:3) consists of CD, TM and SMB1 domains, and truncated 52 (SEQ ID NO:2) only contains the CD domain.

Particularly preferably, the truncated body 144 in engineered exosomes can allow effective and reproducible loading of active bodies, such as therapeutic proteins, into the lumen or extralumen of the exosomes without the need for additional in vitro operations. step.

The object of the present invention is to provide an isolated extracellular vesicle containing a scaffold protein, the vesicle is preferably an exosome, and the scaffold protein is a protein expressed by an exogenous sequence.

The scaffold protein includes an N-terminal cytoplasmic domain (CD), a transmembrane domain (TM), a growth hormone B-like domain 1 (SMB1), a growth hormone B-like domain 2 (SMB2), a phosphodiesterase catalytic domain, One or more of the nuclease-like domains.

In some embodiments, the scaffold protein comprises at least a transmembrane domain (TM) comprising the amino acid sequence VLSLVLSVCVLTTILGCIFGL (SEQ ID NO: 6).

In some embodiments, the scaffold protein comprises at least CD, TM, SMB1 domains.

In some embodiments, the scaffold protein includes at least: CD, TM, SMB1, SMB2, a phosphodiesterase catalytic domain, and a nuclease-like domain;

In some embodiments, the scaffold protein includes at least: CD, TM, SMB1, SMB2, and phosphodiesterase catalytic domains;

In some embodiments, the scaffold protein comprises at least: CD, TM, SMB1 and SMB2 domains;

In some embodiments, involving expression or self-assembly by fusion of scaffold proteins with native full-length proteins or biologically active molecules or fragments (e.g., therapeutically relevant proteins, targeting peptides), the fused or self-assembled proteins are present within EVs A luminal surface or extraluminal method, and EVs containing the protein obtained by this method. Natural full-length proteins or bioactive fragments of bioactive molecules (eg, therapeutically relevant proteins, targeting peptides) can be transported into the lumen or extralumen of EVs by conjugation with scaffold proteins.

Some embodiments of the invention relate to cell engineering methods for the production of engineered EVs, particularly exosomes, by transiently or stably introducing plasmids into Expi293F cells to produce engineered exosomes. In some embodiments, the cell contains a plasmid containing the exogenous sequence.

In embodiments of the present invention, in the engineered EVs, scaffold proteins have a higher density than conventional endogenous proteins. In some embodiments, conventional endogenous EV (eg, exosome) proteins are selected from the following group: PDGFRN, LAM2B, and fragments thereof, with PTGFRN as a positive control.

In some embodiments, exogenous sequences are inserted into the N-terminus or C-terminus of ENPP1, Rab7a, STX7, EPCAM, CXADR.

In some embodiments, the scaffold protein is a fusion protein comprising a scaffold protein, such as ENPP1, Rab7a, STX7, EPCAM, CXADR, or fragments thereof, and a therapeutically active substance, such as an active protein, a therapeutic peptide, a targeting peptide.

In some embodiments, the exogenous sequence encodes a biologically active molecule (eg, therapeutic peptide, targeting peptide). In some embodiments, the therapeutic peptide is an enzyme, ligand, receptor, transcription factor or fragment or modification thereof, an antimicrobial peptide or fragment or modification thereof. The therapeutic peptide is selected from the group consisting of natural peptides, recombinant peptides, synthetic peptides, or linkers attached to the therapeutic compound. In some embodiments, the therapeutic peptide is an antibody or fragment or modification thereof. In specific embodiments, the antibodies are Nanobodies. Antibodies used in EVs of the invention can be any antigen-binding molecule known in the art, including, for example, surrogate antibody formats, antigen-drug conjugates (ADCs), or immunotoxins.

In some embodiments, the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, and small molecules.

In some embodiments, the exogenous sequence encodes a targeting moiety. In some embodiments, the targeting moiety is specific for an organ, tissue or cell.

In some embodiments, the coding sequence is codon optimized.

In another aspect, the present invention also relates to methods of modifying the pharmacokinetic or pharmacodynamic characteristics of small molecule drugs. These methods include loading the small molecule onto EVs and/or binding proteins present in EVs to modulate the in vivo and potentially in vitro properties of the small molecule drug.

On the other hand, the present invention provides a pharmaceutical composition comprising the aforementioned extracellular vesicle and a pharmaceutically acceptable carrier.

The present invention also provides modified cells that produce the above-mentioned extracellular vesicles. Preferably, the cells include a vector, and the vector contains a nucleic acid sequence encoding the aforementioned scaffold protein and bioactive molecule. Preferably, the nucleic acid sequence is operably linked to a promoter.

The present invention also provides a method for anchoring a bioactive molecule to an extracellular vesicle, which includes connecting the bioactive molecule to the aforementioned scaffold protein.

The technical solution of the present invention is achieved through the following steps:

Step One: Identification of High-Density Scaffolding Proteins

Expi293F cell culture medium from suspension culture was processed by filtration and ultracentrifugation to prepare EV (exosome) particles. The extracted exosomes were characterized by WB, NTA and electron microscopy. High-density scaffolding proteins on exosomes were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Step 2: Validation of high-density scaffold proteins

To assess the relative ability of high-density scaffold proteins to guide fusion partners to EVs, scaffold proteins were labeled with FLAG and loaded into the EV lumen with GFP as a surrogate cargo molecule. The Expi293F cell line encoding GFP fusion scaffold protein (scaffold protein cell line) was established by applying antibiotics.

Flow cytometry was used to measure the GFP expression of scaffold protein cell lines to determine the level of cellular GFP, and Western blot (WB) was used to measure the GFP content in total exosomes. Flow cytometry nanoanalyzer (NanoFCM) measures the GFP fluorescence of individual vesicles. High-density scaffold proteins were verified in three ways, and candidate scaffold proteins with higher loading of GFP into the EV cavity were further screened.

Step Three: Validation of Candidate Scaffolding Proteins

Mass spectrometry detected GFP expression in exosomes (engineered exosomes) produced by scaffold protein cell lines, and WB measured the loading level of GFP in each EV. Co-incubation of engineered exosomes with cells determines the time it takes for cells to phagocytose exosomes and the efficiency of phagocytosis.

Step 4: Construct engineered exosomes capable of displaying a variety of bioactive molecules

Through a series of screenings, a scaffold protein better than PTGFRN was obtained, and ENPP1 was used as an example to construct engineered exosomes to display bioactive molecules. According to the domain structure of ENPP1 protein, a truncated body of ENPP1 was constructed to determine the minimum sequence related to EV localization. A plasmid that fuses and expresses bioactive molecules at the C-terminus of ENPP1 is constructed, and engineered exosomes are produced by transiently transfecting Expi293F cells and can display different bioactive molecules on the surface of exosomes. Combine confocal, qPCR experiments and WB experiments to verify the functions of bioactive factors.

Step 5: Verify the function of the optimized scaffold protein to load multiple bioactive molecules

ENPP1 has the ability to load high levels of GFP into the EV lumen, and on this basis, further expands the range of cargo (bioactive substances) types that can be loaded into EVs. The N-terminal fragment is fused to the lumen of the ENPP1 truncated body to load bioactive factors of different sizes and complexities, including but not limited to FGF18, ANGPTL3, CRISPR-Cas13d and CRISPR-Cas9. The function of loading bioactive factors was verified by combining qPCR experiments, RIP experiments and confocal microscopy.

Step 6: Verify the function of engineered exosomes to load a wide range of mRNA categories

RNA-based therapies are very promising for treating a variety of diseases because they can address the genetic origin in a variety of possible ways. However, RNA molecules cannot cross cell membranes, so safe and effective delivery vehicles are crucial. EVs are endogenous nanoscale particles with multiple properties that make them ideal candidates for therapeutic RNA delivery. To this end, the present invention uses the sorting effect of 144 to sort MS2 proteins into exosomes. In addition, the MS2 recognition stem-loop structure loop is placed in the non-coding region of the target mRNA, and the specific binding of RNA and binding proteins is used to realize the mRNA Efficient loading in exosomes.

Description of drawings

Figure 1: Identification of high-density scaffold proteins in exosomes from Expi293F cells;

Figure 2: Verification diagram of high-density scaffold protein effect;

Figure 3: ENPP1 truncated body capacity evaluation chart;

Figure 4: The scaffold protein of the present invention displays a targeting peptide map on the EV surface;

Figure 5: The scaffold protein of the present invention displays a targeting peptide map on the EV surface;

Figure 6: Diagram of the scaffold protein of the present invention loading CRISPR-Cas13d into the EV cavity;

Detailed ways

This invention is not limited to the specific compositions or process steps described. Those skilled in the art will understand upon reading this disclosure that each of the individual aspects described and illustrated herein has discrete components and features that can be separated or combined with the features of any additional aspects without departing from them. the scope or spirit of the disclosure. Any stated method may be performed in the order of events stated or in any other order logically possible.

The specific embodiments of the present invention are not intended to limit the various aspects of the present disclosure, which may be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting, and the scope of the disclosure will be limited only by the appended claims.

In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to examples. The examples are only used to explain the present invention and do not limit the scope of protection. The instruments, equipment, consumables and reagents used are commercially available products unless otherwise specified.

Step one: Obtain and characterize Expi293FEV

1.Extraction of Expi293FEV

Expi293F cells were inoculated into a 250 ml culture flask at 1 × 10 ⁶ cells/ml. When the density reached 4 × 10 ⁶ cells/ml, the supernatant was collected. Centrifuge at 500 g for 10 min at 4°C to remove cell pellets. The supernatant was collected and centrifuged at 3000 g for 20 min at 4°C to remove cell debris. Collect the supernatant, centrifuge at 10000g for 30 minutes at 4°C, collect the supernatant and pass it through a 0.22um filter membrane. The filtered supernatant was adsorbed by Evtrap magnetic beads to obtain EVs.

2. Mass spectrometry method to detect exosome proteins.

An appropriate amount of peptide fragments were taken from each sample for chromatographic separation using a nanoliter flow rate Easy nLC 1200 chromatography system (ThermoScientific). Buffer solution: Solution A is 0.1% formic acid aqueous solution, solution B is 80% ACN/0.1% formic acid. The column was equilibrated with 100% solution A. After injection, the sample was subjected to gradient separation through a chromatographic analysis column with a flow rate of 300 nl/min.

The liquid phase separation gradient is as follows: 0 minutes---3 minutes, linear gradient of liquid B from 2% to 8%; 3 minutes---81 minutes, linear gradient of liquid B from 8% to 40%; 81 minutes---83 Minutes, the linear gradient of liquid B is from 40% to 95%; from 83 minutes to 90 minutes, liquid B is maintained at 95%.

After peptide separation, DDA (data-dependent acquisition) mass spectrometry was performed using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). The analysis time is 90 minutes; the detection mode is: positive ion, precursor ion scanning range: 400-1200m/z, first-level mass spectrometry resolution: 60,000@m/z 200, AGC target: 3e6, first-level Maximum IT: 30ms.

The secondary mass spectrometry analysis of peptides was collected according to the following method: after each full scan (full scan), the secondary mass spectrometry spectrum (MS2scan) of the 20 highest intensity precursor ions was triggered and collected. The secondary mass spectrometry resolution: 30,000@m/z 200, AGC target: 1e5, secondary Maximum IT: 50ms, MS2 Activation Type: HCD, Isolation window: 1.6Th, Normalized collision energy: 28.

Step 2: Screening and verification of scaffold proteins

1. Screening of scaffold proteins

Screen high-density scaffold proteins according to the following criteria: A. High-density proteins in Expi293F exosomes (protein abundance >1×10 ⁷ ); B. Membrane proteins; C. Exclude proteins from existing technologies. A total of 21 proteins were screened: ENPP1, Rab7a, STX7, STX4, EPCAM, CXADR, TRFC, TRFC-81, CD55, IST1, VTA1, SNAP23, AT1A1, PDL1, VAMP2, Sytenin. The above proteins were used for subsequent verification and experiments. The results are shown in Figures 1A and 1B. Figure 1A shows the mass spectrometry identification of proteins in Expi293F exosomes, and the heat map shows the abundance of exosome marker genes; Figure 1B shows the Venn diagram showing the high density of membrane proteins in exosomes.

2. Construction and verification of stably transfected cell lines

The plasmid of the above scaffold protein was constructed and transfected into Expi293F cells by electroporation (celetrix). Construct a stable cell line by supplementing neomycin with regular passages until cell line viability returns to more than 90%. Flow cytometry showed that more than 90% of the cells expressed GFP, which was considered to be a successful stably transduced cell line, as shown in Figure 2A.

3. Characterization of particle size of engineered exosomes

Use Nanocoulter instrument to measure particle size changes of engineered exosomes. Compared with the particle size of Expi293F exosomes, engineered exosomes are more heterogeneous and have a wider particle size distribution, but are concentrated between 60-150nm. See Figure 2B.

4. GFP enrichment of engineered exosomes

Using 1um particle size magnetic beads to capture exosomes for routine flow cytometry analysis, the total fluorescence intensity of exosomes can be obtained. The flow nanoanalyzer (DxFLEX), a cytometer that can analyze particles smaller than visible light wavelengths, can be used to measure the GFP fluorescence of individual exosomes. Detect the expression of GFP in engineered exosomes according to the WB procedure of Example 1.2. Combining the two nanoflow detection protocols and WB results, Rab7a, ENPP1, EPCAM, STX7, CXADR, TRFC, PDL1, SNAP23, Sytenin, VAMP2, VTA1, TRFC-81 and CD55 were confirmed in at least one detection protocol. GFP is abundant in exosomes. See attached Figures 2C and 2D.

5. Calculation of engineered exosome capacity

The His standard curve was drawn by WB method and the vector of engineered exosomes was calculated. Briefly, the protein lysate of exosomes (1×10 ¹⁰ ) and His recombinant protein (25ng, 50ng, 100ng, 200ng, 400ng, 800ng and 1600ng) were loaded to detect the protein expression of Flag and His. Next, the expression of GFP in engineered exosomes was detected by mass spectrometry. First, calculate the gray value of His protein through ImageJ and draw a standard curve, R2 = 0.9716. Secondly, the number of Flags loaded on each exosome is calculated based on the gray value of the engineered exosomes combined with the number of exosomes. As shown in Figure 2G , the engineered exosome loads overexpressed by Rab7a (~600/EV) and ENPP1 (~500/EV) were higher than those of PTGFRN (~400/EV). Furthermore, the mass spectrometry method also showed that the loading of Rab7a and ENPP1 was higher than that of PTGFRN. Therefore, both ENPP1 and Rab7a were further evaluated as scaffolds for the display of proteins of interest in EVs. The above experimental results are shown in Figures 2E-H.

Step 3: Efficiency of engineered exosomes taken up by cells

1. Co-incubation of engineered exosomes with cells

100w Expi293F cells were seeded in each well in a 6-well plate, and 1× ¹⁰ engineered exosomes (exosomes derived from ENPP1, Rab7a and PTGFRN) were incubated with the cells. The cells were collected after 24 hours and centrifuged at 1000 rpm for 5 min. , discard the supernatant, wash twice with PBS, collect the cell pellet after centrifugation, and detect intracellular Flag expression according to the WB method. See Figure 2I. From the results, it can be seen that although the Rab7a-derived engineered exosomes have a high GFP loading, they are not easily phagocytosed by cells, while the ENPP1-derived engineered exosomes are phagocytosed and internalized by cells much more efficiently than PTGFRN. source of engineered exosomes. In summary, ENPP1-derived engineered exosomes are superior to PTGERN-derived exosomes in terms of content loading and ability to be phagocytized and internalized by cells.

Step 4: ENPP1 can display multiple proteins on EV surface

1.Evaluation of ENPP1 truncated body capacity

ENPP1 is a protein containing 925 amino acids, which contains N-terminal cytoplasmic domain (CD), transmembrane domain (TM), growth hormone B-like domain 1 (SMB1), growth hormone B-like domain 2 (SMB2) , phosphodiesterase catalytic domain, nuclease-like domain. Truncate the ENPP1 protein domain, load EGFP at the N-terminus of the truncated body or full-length (FL, SEQ ID NO: 1) and add a Flag tag at the C-terminus, and construct a plasmid based on this structure. Specifically, truncated form 596 (SEQ ID NO:5) contains CD, TM, SMB1, SMB2, and phosphodiesterase catalytic domains. Truncated 190 (SEQ ID NO:4) contains CD, TM, SMB1 and SMB2 domains. Truncated 144 (SEQ ID NO:3) consists of CD, TM and SMB1 domains, and truncated 52 (SEQ ID NO:2) only contains the CD domain. Cells of full-length ENPP1 and ENPP1 truncated forms were obtained by transient transfection, and the expression of GFP in the cells was evaluated by flow cytometry. The supernatant was collected for 48 hours and exosomes were extracted. The exosomes were counted using the Nanocoulter instrument, and the expression of Flag was detected by WB method to evaluate the EGFP loading of ENPP1 full-length and truncated bodies. Flow cytometry results showed that although the transfection efficiency of truncated body 52 was high, its exosome loading efficiency was very low. Further research found that the expression of ENPP1 in exosomes will be significantly reduced once the TM and SMB1 domains are deleted. This result suggests that this domain is an essential structure for the localization of ENPP1 in exosomes. In addition, compared with FL, the expression of truncated body 596 in exosomes was significantly reduced, suggesting that the nuclease-like domain is also involved in the localization of ENPP1 in exosomes. According to the WB gray value calculation, the number of 144 exosomes loaded with Flag is about 600, and the number of ENPP1 full-length exosomes loaded with is about 200. See Figure 3.

2. ENPP1 and truncated bodies loaded with targeting peptides

Six plasmids were constructed, encoding ENPP1-CAP (chondrocyte affinity peptide), 144-CAP, EPCAM-CAP, STX7-CAP, CXADR-CAP and PTGFRN-CAP respectively. The ENPP1-CAP plasmid contains a glycosylation sequence (GNSTM), a CAP sequence (DWRVIIPPRPSA) and a glycine-serine spacer located at the C-terminus of the ENPP1 protein. Other plasmid construction methods are the same as above. The above plasmids were transiently transfected to produce exosomes containing CAP peptide. The exosomes of CAP peptide were incubated with 1mM DiI dye at a volume ratio of 400:1 for 30 minutes at 37° in the dark. The free unbound dye was removed by a 0.22um filter to obtain DiI-labeled exosomes.

Spread the cell sheets at the bottom of the 12-well plate, add 8w rat chondrocytes to each well, and let stand overnight in a 37°C incubator. Add 20ug of labeled material into a 12-well plate, incubate for a total of 3 hours, and discard the cell culture medium. Wash 3 times with PBS and fix with 4% paraformaldehyde for 20 min. Wash 3 times with PBS, add 500ul DMEM and drop a drop of Hochest stain, and incubate at 37°C for 30 minutes. Put a drop of anti-quenching reagent on the glass slide, fix the slide, and use confocal FV1000 to detect the efficiency of exosome uptake by cells. The results are shown in Figures 4 and 5. A significant DiI signal was observed in the experimental group expressing the subject scaffold protein fused with CAP, and the signal was significantly greater than the empty exosome control group, indicating that the CAP peptide promoted the entry of exosomes into chondrocytes. In addition, confocal results demonstrated that EPCAM, STX7, CXADR, ENPP1 and truncated 144 scaffolds can effectively display functional proteins on the EV surface.

Step 5: ENPP1 enables EV luminal loading of a broad class of proteins

1. FL ENPP1 and truncated 144 scaffolds load CRISPR-Cas13d

CRISPR-Cas13d was constructed at the N-terminus of ENPP1 and a truncated 144 scaffold and transiently transduced to generate luminal CRISPR-Cas13d-enriched exosomes. The expression of Cas13d and sgRNA in cells and exosomes was detected by real-time fluorescence quantitative PCR. See Figures 6A-D. Figures 6A-B show the construction of ENPP1-Cas13d fusion plasmid and 144-Cas13d fusion plasmid, and detect the expression of cas13d and sgRNA in cells respectively; Figure 6C-D show the detection of cas13d and sgRNA respectively in exosomes. Expression of sgRNA, where NC is empty exosomes.

Real-time fluorescence quantitative PCR (qRT-PCR): Use Vazyme's reverse transcription kit HiScript II Q RTSuperMix for qPCR (+gDNA wiper) (R223-01) and qPCR kit AceQ Universal SYBR qPCRMaster Mix (Q511-02). The relevant primers involved in the experiment were synthesized by General Biotechnology (Anhui) Co., Ltd.

sgRNA:sense:CACCGAACCCTAACCAAC,

antisense:TGCTGTTTCAAACCCCGAC;

Cas13d: sense:AGCTGACCAACTCCTTCTCC,

antisense:GCATCACTTCCCTGAGCTTG;

GAPDH: sense:AGACAGCCGCATCTTCTTGT,

antisense:CTTGCCGTGGGTAGAGTCAT.

2. Recipient cells take up exosomal sgRNA and Cas13d protein

CRISPR-Cas13d-loaded exosomes, empty exosomes (NC) and empty exosomes (NC) (whether correct) were co-incubated with 293T cells. The cells were collected at 48 hours and the expression of Cas13d and sgRNA in the cells was detected. See Figure 6E-F.

Compared with empty exosomes, ENPP1 and truncated 144 scaffold exosomes were co-incubated with cells, which increased the cas13d content in cells by 6.13 and 6.82 times, respectively, and the sgRNA content by 3.72 and 3.15 times.

Claims (42)

1. An isolated extracellular vesicle containing a scaffold protein, characterized in that the scaffold protein is a protein expressed from an exogenous sequence, and the scaffold protein includes an N-terminal cytoplasmic domain (CD), a transmembrane domain (TM) ), one or more of growth hormone B-like domain 1 (SMB1), growth hormone B-like domain 2 (SMB2), phosphodiesterase catalytic domain, and nuclease-like domain. 2. The extracellular vesicle of claim 1, wherein the scaffold protein has a density higher than conventional endogenous proteins of the extracellular vesicle. 3. The extracellular vesicle of claim 1, wherein the scaffold protein at least contains a transmembrane region. 4. The extracellular vesicle of claim 3, wherein the membrane-penetrating region comprises the VLSLVLSVCVLTTILGCIFGL amino acid sequence. 5. The extracellular vesicle of claim 3, wherein the scaffold protein at least includes an N-terminal cytoplasmic domain, a transmembrane region, and a growth hormone B-like domain 1; or at least includes: an N-terminal cytoplasmic domain. Domain, transmembrane region, growth hormone B-like domain 1 and growth hormone B-like domain 2 domains; or at least comprising: N-terminal cytoplasmic domain, transmembrane region, growth hormone B-like domain 1, growth hormone B-like Domain 2 and phosphodiesterase catalytic domain; or at least include: N-terminal cytoplasmic domain, transmembrane region, growth hormone B-like domain 1, growth hormone B-like domain 2, phosphodiesterase catalytic domain, nuclease domain-like structure. 6. The extracellular vesicle of claim 1, wherein the N-terminal cytoplasmic domain, transmembrane region, growth hormone B-like domain 1, growth hormone B-like domain 2, and phosphodiesterase The catalytic domain and nuclease-like domain are connected via a linker. 7. The extracellular vesicle of claim 6, wherein the linker comprises a peptide bond or one or more amino acids. 8. The extracellular vesicle according to any one of claims 1 to 7, wherein the scaffold protein is selected from the group consisting of ENPP1, Rab7a, STX7, STX4, EPCAM, CXADR, TRFC, TRFC-81, CD55, IST1 , VTA1, SNAP23, AT1A1, PDL1, VAMP2, Sytenin or fragments, variants, derivatives or modifications thereof. 9. The extracellular vesicle of any one of claims 1-8, wherein the length of the scaffold protein is at least about 52, at least about 62, at least about 76, at least about 77, at least About 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least About 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least About 100, at least about 105, at least about 110, at least about 115, at least about 120, at least about 125, at least about 130, at least about 135, at least about 140, at least about 144, at least About 154, at least about 164, at least about 170, at least about 180, at least about 190, at least about 290, at least about 390, at least about 490, at least about 596, at least about 690, at least About 790, at least about 890 or at least about 925 amino acids. 10. The extracellular vesicle of claim 8, wherein the scaffold protein has at least about 50% and at least about 60% of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. , at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or an amino acid sequence with approximately 100% sequence identity. 11. The extracellular vesicle of any one of claims 1-10, comprising a bioactive molecule linked to a scaffold protein. 12. The extracellular vesicle of claim 11, wherein the bioactive molecule is on the lumen surface, on the lumen or on the lumen surface of the extracellular vesicle. 13. The extracellular vesicle of claim 11, wherein the scaffold protein and a bioactive molecule constitute a fusion protein, and the bioactive molecule is an active protein, a therapeutic peptide or a targeting peptide. 14. The extracellular vesicle of claim 13, wherein the bioactive molecule is exogenously encoded. 15. The extracellular vesicle of claim 11, wherein the bioactive molecule is connected to the scaffold protein through a linker. 16. The extracellular vesicle of claim 15, wherein the linker comprises a peptide bond or one or more amino acids. 17. The extracellular vesicle of any one of claims 15-16, wherein the linker includes a cleavable linker or a flexible linker. 18. The extracellular vesicle of claim 11, wherein the biologically active molecules include proteins, polypeptides, peptides, polynucleotides, compounds, viruses, toxins, toxoids, non-toxic mutants of toxins, An ionophore, a carrier for an ionophore, a channel or pore forming moiety, an activator of a positive costimulatory molecule, an activator of a binding partner of a positive costimulatory molecule, or any combination thereof. 19. The extracellular vesicle of claim 18, wherein the protein includes an immunogenic protein, a recombinant peptide, a natural peptide, a synthetic peptide, an antibody, a fusion protein, an enzyme, a cytokine, a ligand, a receptor, or an immunogenic protein. body, transcription factors, T cell receptor (TCR), T cell co-receptor, major histocompatibility complex (MHC), human leukocyte antigen (HLA) and its derivatives, tumor antigens, CRIPSR family proteins, DNA Or RNA editing protein complexes, RNA and DNA binding proteins, interacting proteins, or any combination of the above. 20. The extracellular vesicle of claim 18, wherein the virus includes adeno-associated virus, parvovirus, retrovirus, adenovirus, or any combination thereof. 21. The extracellular vesicle of claim 11, wherein the bioactive molecule is an inhibitor of a negative checkpoint modulator or an inhibitor of a binding partner of a negative checkpoint modulator. 22. The extracellular vesicle of claim 21, wherein the negative checkpoint modulator is selected from the group consisting of: cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed cell death Protein 1 (PD-1), lymphocyte activating gene 3 (LAG-3), T cell immunoglobulin mucin-containing protein 3 (TIM-3), B and T lymphocyte attenuation factor (BTLA), with Ig and ITIM domain T cell immunoreceptor (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indole Amine 2,3-dioxygenase (IDO), CD20, CD39 and CD73. 23. The extracellular vesicle of claim 18, wherein the toxin is diphtheria toxin. 24. The extracellular vesicle of claim 18, wherein the toxoid is tetanus toxoid. 25. The extracellular vesicle of claim 18, wherein the atoxic mutant of the toxin is a nontoxic mutant of diphtheria toxin. 26. The extracellular vesicle of claim 18, wherein the positive costimulatory molecule is a member of the TNF receptor superfamily. 27. The extracellular vesicle of claim 26, wherein the TNF receptor superfamily member is selected from the group consisting of: CD120a, CD120b, CD18, OX40, CD40, Fas receptor, M68, CD27 , CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, RANK, OCIF, TWEAK receptor, TACI, BAFF receptor, ATAR, CD271, CD269, AITR, TROY, CD358, TRAMP and XEDAR. 28. The extracellular vesicle of claim 18, wherein the activator of the positive costimulatory molecule is a member of the TNF superfamily. 29. The extracellular vesicle of claim 28, wherein the TNF superfamily member is selected from the group consisting of: TNFα, TNF-C, OX40L, CD40L, FasL, LIGHT, TL1A, CD27L, Siva , CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand and EDA-2. 30. The extracellular vesicle of claim 18, wherein the positive costimulatory molecule is a CD28 superfamily costimulatory molecule. 31. The extracellular vesicle of claim 30, wherein the CD28 superfamily costimulatory molecule is ICOS or CD28. 32. The extracellular vesicle of claim 18, wherein the activator of the positive costimulatory molecule is ICOSL, CD80 or CD86. 33. The extracellular vesicle of claim 18, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-10, IL-12 and IL-15. 34. The extracellular vesicle of claim 19, wherein the tumor antigen is selected from the group consisting of alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), Mucin 1 (MUC1), Tn-MUC1, Mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, program Sexual death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, α - Folate receptor, CE7R, IL-3, cancer-testis antigen, MART-1gp100 and TNF-related apoptosis-inducing ligand. 35. The extracellular vesicle of claim 19, wherein the CRIPSR family protein includes Cas9 or Cas13. 36. The extracellular vesicle of claim 18, wherein the polynucleotide comprises a DNA or RNA aptamer. 37. The extracellular vesicle of any one of claims 1-36, wherein the extracellular vesicle is an exosome, a small vesicle or a large vesicle. 38. A pharmaceutical composition comprising the extracellular vesicle of any one of claims 1-37 and a pharmaceutically acceptable carrier. 39. A cell producing the extracellular vesicle of any one of claims 1-37. 40. The cell of claim 39, comprising a vector comprising a nucleic acid sequence encoding the scaffold protein and/or biologically active molecule of any one of claims 1-37. 41. The cell of claim 40, wherein the nucleic acid sequence is operably linked to a promoter. 42. A method of anchoring a bioactive molecule to an extracellular vesicle, comprising connecting the bioactive molecule to the scaffold protein of any one of claims 1-37.