WO2024216978A1 - Engineered extracellular vesicle and use thereof - Google Patents

Abstract

Provided is a method for loading a bioactive substance in an extracellular vesicle, particularly in an exosome. The method comprises: producing a lumen engineered exosome, the lumen engineered exosome comprising one or more exosome proteins of high concentration, a modification or fragment of the exosome protein, or a fusion protein of the exosome protein and a therapeutic or cargo protein. A preparation method comprises: producing an engineered exosome, the exosome comprising one or more of a protein having a higher concentration than that observed in a wild-type exosome, a modification or fragment of the protein or a fusion protein of the protein and a payload.

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 for preventing or treating cancer and other diseases.

Background Art

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. Exosomes, as a new treatment method, provide many advantages over conventional drug delivery methods in many therapeutic areas.

The main feature of exosomes is the ability to contain biologically active payloads in their internal space or cavity. It is well known that exosomes contain endogenous payloads, including mRNA, miRNA, DNA, proteins, carbohydrates and lipids, but the ability to specifically load the desired therapeutic payload is currently limited. Exosomes can be loaded by overexpressing the desired therapeutic payload in production cells, but the efficiency of this loading is generally limited because the payload is randomly located to the exosome processing center of the cell. In addition, purified exosomes such as siRNA can be loaded in vitro by, for example, electroporation. The efficiency of these methods may be low or limited to small payloads. Therefore, the generation of efficient and well-defined loaded exosomes can better achieve therapeutic uses and other applications based on exosome technology.

Summary of the invention

There are many conventional endogenous proteins in extracellular vesicles (EVs), such as exosomes, small vesicles, and large vesicles. Taking exosomes as an example, these proteins can regulate the fusion of exosome membranes with receptor cells, exosome membrane exchange, and fusion, etc. The inventors screened and identified a group of proteins on the surface of exosome membranes, overexpressed the required proteins in production cells, and enriched these proteins in exosomes as scaffold proteins, especially on the exosome membranes, with significantly higher concentrations than 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 scaffold proteins, ENPP1, Rab7a, EPCAM, STX7, and CXADR were shown to have preferred effects, especially ENPP1, which had good effects in all aspects, including excellent loading level, shorter cell phagocytosis time and phagocytosis efficiency.

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

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

The truncations were performed according to the domain structure of the ENPP1 protein. Specifically, truncation 596 (SEQ ID NO: 5) contained CD, TM, SMB1, SMB2, and phosphodiesterase catalytic domains. Truncation 190 (SEQ ID NO: 4) contained CD, TM, SMB1, and SMB2 domains. Truncation 144 (SEQ ID NO: 3) consisted of CD, TM, and SMB1 domains, and truncation 52 (SEQ ID NO: 2) contained only the CD domain.

Particularly preferably, the truncated body 144 in the engineered exosomes can allow efficient and reproducible loading of active substances, such as therapeutic proteins, into the lumen or outside the lumen of the exosomes without the need for additional in vitro manipulation steps.

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

The scaffold protein comprises one or more of an N-terminal cytoplasmic domain (CD), a transmembrane region (TM), a growth hormone B-like domain 1 (SMB1), a growth hormone B-like domain 2 (SMB2), a phosphodiesterase catalytic domain, and a nuclease-like domain.

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

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

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

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

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

In some embodiments, the present invention relates to a method of expressing or self-assembling a scaffold protein by fusing it with a natural full-length protein or a biologically active molecule or fragment (e.g., a therapeutically relevant protein, a targeting peptide), so that the fused or self-assembled protein is present on the surface of the luminal cavity of an EV or outside the cavity, and an EV containing the protein obtained by the method. The biologically active fragment of a natural full-length protein or a biologically active molecule (e.g., a therapeutically relevant protein, a targeting peptide) can be transported to the lumen or outside the lumen of an EV by conjugating it to a scaffold protein.

Some embodiments of the present invention relate to a cell engineering method for producing engineered EVs, especially exosomes, by transiently or stably introducing a plasmid into Expi293F cells to produce engineered exosomes. In some embodiments, the cell comprises a plasmid containing an exogenous sequence.

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

In some embodiments, the exogenous sequence is 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 a fragment thereof, and a therapeutically active substance, such as an active protein, a therapeutic peptide, or a targeting peptide.

In some embodiments, the exogenous sequence encodes a biologically active molecule (e.g., a therapeutic peptide, a targeting peptide). In some embodiments, the therapeutic peptide is an enzyme, a ligand, a receptor, a transcription factor, or a fragment or modification thereof, an antimicrobial peptide, or a fragment or modification thereof. The therapeutic peptide is selected from the group consisting of: a natural peptide, a recombinant peptide, a synthetic peptide, or a linker connected to a therapeutic compound. In some embodiments, the therapeutic peptide is an antibody, or a fragment or modification thereof. In a specific embodiment, the antibody is a nanobody. The antibody used in the EV of the present invention can be any antigen binding molecule known in the art, including, for example, alternative antibody forms, 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 to an organ, tissue or cell.

In some embodiments, the coding sequence is codon optimized.

In another aspect, the present invention also relates to methods for altering 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 potential in vitro properties of the small molecule drug.

In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned extracellular vesicles and a pharmaceutically acceptable carrier.

The present invention also provides a modified cell for producing the above-mentioned extracellular vesicles, preferably, the cell comprises a vector, the vector comprises a nucleic acid sequence encoding the above-mentioned scaffold protein and biologically active molecule. Preferably, the nucleic acid sequence is operably linked to a promoter.

The present invention also provides a method for anchoring a biologically active molecule to an extracellular vesicle, comprising connecting the biologically active molecule to the aforementioned scaffold protein.

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

Step 1: Identification of high-density scaffold 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. Scaffold proteins with high density on exosomes were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Step 2: Validation of high-density scaffold protein

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

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

Step 3: Validation of candidate scaffold proteins

Mass spectrometry was used to detect GFP expression in exosomes produced by the scaffold protein cell line (engineered exosomes), and WB was used to measure the GFP loading level in each EV. Engineered exosomes were co-incubated with cells to determine the time and efficiency of cell phagocytosis of exosomes.

Step 4: Constructing engineered exosomes capable of displaying multiple bioactive molecules

Through a series of screening and verification, a scaffold protein that is better than PTGFRN was obtained, and ENPP1 was used as an example to construct engineered exosomes to display bioactive molecules. According to the structural domain of ENPP1 protein, a truncated version of ENPP1 was constructed to determine the minimum sequence related to EV localization. A plasmid was constructed to express bioactive molecules fused to the C-terminus of ENPP1, and Expi293F cells were transiently transfected to produce engineered exosomes and display different bioactive molecules on the surface of exosomes. The functions of bioactive factors were verified by combining confocal, qPCR and WB experiments.

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

ENPP1 has the ability to load high levels of GFP into the EV lumen, on this basis, further expanding the range of cargo (bioactive substances) that can be loaded into EVs. By fusing to the N-terminal fragment of the ENPP1 truncated lumen to load bioactive factors of different sizes and complexities, including but not limited to FGF18, ANGPTL3, CRISPR-Cas13d and CRISPR-Cas9. Combining qPCR experiments, RIP experiments and confocal microscopy to verify the function of loaded bioactive factors.

Step 6: Validate the functionality of engineered exosomes loaded with a broad range of mRNAs

RNA-based therapies are very promising for treating a variety of diseases because they can address the genetic origin issue in a variety of possible ways. However, RNA molecules cannot cross cell membranes, so safe and effective delivery vehicles are essential. EVs are endogenous nanoparticles with a variety of properties that make them ideal candidates for therapeutic RNA delivery agents. To this end, the present invention utilizes 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 to the binding protein is utilized to achieve efficient loading of mRNA in exosomes.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 2: Validation of the effect of high-density scaffold protein;

Figure 3: Evaluation of the loading capacity of ENPP1 truncated bodies;

Figure 4: Targeted peptide map displayed on the surface of EV by the scaffold protein of the present invention;

Figure 5: Targeted peptides displayed on the surface of EV by the scaffold protein of the present invention;

Figure 6: A diagram of the scaffold protein of the present invention loading CRISPR-Cas13d in the EV cavity;

DETAILED DESCRIPTION

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

The specific embodiments of the present invention are not limitations on the various aspects of the present disclosure, and the various aspects can be defined by reference to the entire specification. It should also be understood that the terms used in the present invention are only used for the purpose of describing specific aspects and are not intended to be restrictive, and the scope of the present disclosure will only be limited by the attached claims.

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

Step 1: Acquisition and characterization of Expi293FEV

1. Extraction of Expi293FEV

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

2. Detection of exosomal proteins by mass spectrometry.

An appropriate amount of peptides was taken from each sample and chromatographically separated using the nanoliter flow rate Easy nLC 1200 chromatography system (Thermo Scientific). Buffer: Solution A is 0.1% formic acid in water, and solution B is 80% ACN/0.1% formic acid. The chromatographic column was balanced with 100% solution A. After sample injection, gradient separation was performed on the chromatographic analysis column at a flow rate of 300nl/min.

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

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

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

Step 2: Screening and verification of scaffold proteins

1. Screening of Scaffold Proteins

High-density scaffold proteins were screened according to the following criteria: A. Proteins with high density in Expi293F exosomes (protein abundance>1×10 ⁷ ); B. Membrane proteins; C. Proteins that exclude prior art. 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. The experimental results are shown in Figures 1A and 1B, where Figure 1A is a mass spectrometry identification of proteins in Expi293F exosomes, and the heat map shows the abundance of exosome marker genes; Figure 1B is a Venn diagram showing high-density membrane proteins in exosomes.

2. Construction and verification of stable cell lines

The plasmid of the above scaffold protein was constructed and transfected into Expi293F cells by electroporation (celetrix). Stable cell lines were constructed by regular subculturing and supplementation with neomycin until the cell line viability recovered to more than 90%. When more than 90% of the cells expressed GFP by flow cytometry, it was considered that the stable cell line was successfully constructed, as shown in Figure 2A.

3. Characterization of the Particle Size of Engineered Exosomes

The size variation of engineered exosomes was measured using a Nanocoulter instrument. Compared with the size of Expi293F exosomes, engineered exosomes have higher heterogeneity and a wider range of particle size distribution, but are concentrated between 60-150nm. See Figure 2B.

4. GFP Enrichment of Engineered Exosomes

Using magnetic beads with a particle size of 1um to capture exosomes for conventional flow analysis, the total fluorescence intensity of exosomes can be obtained. The flow nanoanalyzer (DxFLEX) can analyze cytometry of particles smaller than the wavelength of visible light, which can be used to measure the GFP fluorescence of a single exosome. The expression of engineered exosome GFP was detected according to the WB operation steps of Example 1.2. Combining the two nanoflow detection schemes and WB results, Rab7a, ENPP1, EPCAM, STX7, CXADR, TRFC, PDL1, SNAP23, Sytenin, VAMP2, VTA1, TRFC-81 and CD55 confirmed the presence of abundant GFP in their exosomes in at least one detection scheme. See Figures 2C and 2D.

5. Calculation of the amount of engineered exosomes

The His standard curve was drawn by WB method and the carrier of engineered exosomes was calculated. In brief, protein lysate of exosomes (1×10 ¹⁰ ), 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, the gray value of His protein was calculated by ImageJ and the standard curve was drawn, R2=0.9716. Secondly, the number of Flags loaded per exosome was calculated based on the gray value of the engineered exosomes combined with the number of exosomes. As shown in Figure 2G, the loading capacity of engineered exosomes overexpressing Rab7a (~600/EV) and ENPP1 (~500/EV) is higher than that of PTGFRN (~400/EV). Furthermore, mass spectrometry 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 displaying proteins of interest in EVs. The above experimental results are shown in Figures 2E-H.

Step 3: Efficiency of engineered exosomes uptake by cells

1. Co-incubation of engineered exosomes with cells

100w Expi293F cells were inoculated in each well of a 6-well plate, and 1× ¹⁰¹⁰ engineered exosomes (exosomes derived from ENPP1, Rab7a, and PTGFRN) were co-incubated with the cells. After 24 hours, the cells were collected and centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS. After centrifugation, the cell pellet was obtained and the Flag expression in the cells was detected by WB method. See Figure 2I, The results show that although the GFP loading capacity of Rab7a-derived engineered exosomes is high, they are not easily phagocytosed by cells, while the efficiency of ENPP1-derived engineered exosomes being phagocytosed and internalized by cells is much higher than that of PTGFRN-derived engineered exosomes. In summary, whether it is the content loading capacity or the ability to be phagocytosed and internalized by cells, ENPP1-derived engineered exosomes are superior to PTGERN-derived exosomes.

Step 4: ENPP1 can display multiple proteins on the surface of EVs

1. Evaluation of ENPP1 truncated body loading

ENPP1 is a protein containing 925 amino acids, which includes an N-terminal cytoplasmic domain (CD), a transmembrane region (TM), a growth hormone B-like domain 1 (SMB1), a growth hormone B-like domain 2 (SMB2), a phosphodiesterase catalytic domain, and a nuclease-like domain. According to the domain structure of the ENPP1 protein, truncations were performed, EGFP was loaded at the N-terminus of the truncated or full-length (FL, SEQ ID NO:1), and a Flag tag was added to the C-terminus, and plasmids were constructed according to this structure. Specifically, truncation 596 (SEQ ID NO:5) contains CD, TM, SMB1, SMB2, and a phosphodiesterase catalytic domain. Truncation 190 (SEQ ID NO:4) contains CD, TM, SMB1, and SMB2 domains. Truncation 144 (SEQ ID NO:3) consists of CD, TM, and SMB1 domains, and truncation 52 (SEQ ID NO:2) contains only the CD domain. Cells expressing full-length ENPP1 and truncated ENPP1 were obtained by transient transfection, and the expression of GFP in the cells was evaluated by flow cytometry. After collecting the supernatant for 48 hours to extract exosomes, the exosomes were counted by Nanocoulter instrument, and the expression of Flag was detected by WB method to evaluate the EGFP loading of full-length and truncated ENPP1. The flow cytometry results showed that although the transfection efficiency of truncated 52 was high, its exosome loading efficiency was very low. Further studies found that once the TM and SMB1 domains were missing, the expression of ENPP1 in exosomes would be greatly reduced. 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 596 in exosomes was greatly reduced, suggesting that the nuclease-like domain is also involved in the localization of ENPP1 in exosomes. According to the WB grayscale value, the number of exosomes loaded with Flag of 144 was about 600, and the number of exosomes loaded with full-length ENPP1 was about 200. See Figure 3.

2. ENPP1 and truncated ENPP1 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. The construction methods of other plasmids are the same as above. The above plasmids were transiently transfected to produce exosomes containing CAP peptides. The exosomes of CAP peptides were incubated with 1mM DiI dye at a volume ratio of 400:1 at 37° in the dark for 30min, and the free unbound dye was removed by a 0.22um filter membrane to obtain DiI-labeled exosomes.

The cell slide was laid on the bottom of the 12-well plate, and 8w rat chondrocytes were added to each well and incubated overnight at 37℃. Take 20ug of the labeled cells and add them to the 12-well plate, incubate for 3h, and discard the cell culture medium. Wash 3 times with PBS and fix with 4% paraformaldehyde for 20min. Wash 3 times with PBS, add 500ul DMEM and drop a drop of Hochest staining, and incubate at 37℃ for 30min. Drop a drop of anti-quenching reagent on the slide, fix the slide, and use confocal FV1000 to detect the efficiency of cell uptake of exosomes. The results are shown in Figures 4 and 5. The experimental group with CAP expression fused to the object scaffold protein can observe a significant DiI signal, which is significantly greater than the empty exosome control group, indicating that CAP peptide promotes the entry of exosomes into chondrocytes. In addition, the confocal results prove that EPCAM, STX7, CXADR, ENPP1 and truncated 144 scaffolds can effectively display functional proteins on the surface of EVs.

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

1. FL ENPP1 and truncated 144 scaffold loading CRISPR-Cas13d

CRISPR-Cas13d was constructed at the N-terminus of ENPP1 and truncated 144 scaffolds, and transient transfection was performed to produce exosomes rich in CRISPR-Cas13d in the lumen. The expression of Cas13d and sgRNA in cells and exosomes was detected by real-time fluorescence quantitative PCR. See Figures 6A-D, wherein Figures 6A-B construct ENPP1-Cas13d fusion plasmids and 144-Cas13d fusion plasmids, and the expression of cas13d and sgRNA was detected in cells respectively; Figures 6C-D are for detecting the expression of cas13d and sgRNA in exosomes, respectively, where NC is an empty exosome.

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

sgRNA: sense:CACCGAACCCCTACCAAC, antisense:TGCTGTTTCAAACCCCGAC;

Cas13d: sense:AGCTGACCAACTCCTTCTCC, antisense:GCATCACTTCCCTGAGCTTG;

GAPDH: sense: AGACAGCCGCATCTTCTTGT, antisense: CTTGCCGTGGGTAGAGTCAT.

2. Recipient cells take up exosomal sgRNA and Cas13d protein

The exosomes loaded with CRISPR-Cas13d, empty exosomes (NC) and empty exosomes (NC) (correct) were co-incubated with 293T cells, and the cells were collected after 48 hours to detect the expression of Cas13d and sgRNA in the cells. See Figures 6E-F.

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

Claims (42)

A separated extracellular vesicle containing a scaffold protein, characterized in that the scaffold protein is a protein expressed by an exogenous sequence, and the scaffold protein comprises one or more of an N-terminal cytoplasmic domain (CD), a transmembrane region (TM), a growth hormone B-like domain 1 (SMB1), a growth hormone B-like domain 2 (SMB2), a phosphodiesterase catalytic domain, and a nuclease-like domain. The extracellular vesicle of claim 1, wherein the density of the scaffold protein is higher than that of the conventional endogenous protein of the extracellular vesicle. The extracellular vesicle according to claim 1, characterized in that the scaffold protein comprises at least a transmembrane region. The extracellular vesicle according to claim 3, characterized in that the transmembrane region comprises the amino acid sequence VLSLVLSVCVLTTILGCIFGL. The extracellular vesicle according to claim 3, characterized in that the scaffold protein comprises at least an N-terminal cytoplasmic domain, a transmembrane region, and a growth hormone B-like domain 1; or at least comprises: an N-terminal cytoplasmic domain, a transmembrane region, a growth hormone B-like domain 1, and a growth hormone B-like domain 2 domain; or at least comprises: an N-terminal cytoplasmic domain, a transmembrane region, a growth hormone B-like domain 1, a growth hormone B-like domain 2, and a phosphodiesterase catalytic domain; or at least comprises: an N-terminal cytoplasmic domain, a transmembrane region, a growth hormone B-like domain 1, a growth hormone B-like domain 2, a phosphodiesterase catalytic domain, and a nuclease-like domain. The extracellular vesicle according to claim 1, characterized in that the N-terminal cytoplasmic domain, the transmembrane region, the growth hormone B-like domain 1, the growth hormone B-like domain 2, the phosphodiesterase catalytic domain, and the nuclease-like domain are connected by a linker. The extracellular vesicle of claim 6, wherein the linker comprises a peptide bond or one or more amino acids. The extracellular vesicle according to any one of claims 1 to 7, characterized in that the scaffold protein is selected from ENPP1, Rab7a, STX7, STX4, EPCAM, CXADR, TRFC, TRFC-81, CD55, IST1, VTA1, SNAP23, AT1A1, PDL1, VAMP2, Sytenin or a fragment, variant, derivative or modification thereof. The extracellular vesicle of any one of claims 1 to 8, characterized in that 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. The extracellular vesicle as described in claim 8 is characterized in that the scaffold protein has an amino acid sequence that is at least about 50%, at least about 60%, 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 about 100% identical to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. The extracellular vesicle according to any one of claims 1 to 10, characterized in that it comprises a biologically active molecule connected to a scaffold protein. The extracellular vesicle of claim 11, wherein the biologically active molecule is on the luminal surface, on the lumen, or on the exoluminal surface of the extracellular vesicle. The extracellular vesicle according to claim 11, characterized in that the scaffold protein and the biologically active molecule constitute a fusion protein, and the biologically active molecule is an active protein, a therapeutic peptide or a targeting peptide. The extracellular vesicle of claim 13, wherein the biologically active molecule is exogenously encoded. The extracellular vesicle according to claim 11, characterized in that the biologically active molecule is connected to the scaffold protein via a linker. The extracellular vesicle of claim 15, wherein the linker comprises a peptide bond or one or more amino acids. The extracellular vesicle according to any one of claims 15 to 16, wherein the linker comprises a cleavable linker or a flexible linker. The extracellular vesicle of claim 11, characterized in that the biologically active molecule comprises a protein, a polypeptide, a peptide, a polynucleotide, a compound, a virus, a toxin, a toxoid, a non-toxic mutant of a toxin, an ionophore, a carrier of an ionophore, a portion forming a channel or a pore, an activator of a positive co-stimulatory molecule, an activator of a binding partner of a positive co-stimulatory molecule, or any combination thereof. The extracellular vesicle of claim 18, characterized in that the protein comprises 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, a transcription factor, a T cell receptor (TCR), a T cell co-receptor, a major histocompatibility complex (MHC), a human leukocyte antigen (HLA) and its derivatives, a tumor antigen, a protein within the CRIPSR family, a DNA or RNA editing protein complex, an RNA and DNA binding protein, an interacting protein, or any combination of the above substances. The extracellular vesicle of claim 18, wherein the virus comprises an adeno-associated virus, a parvovirus, a retrovirus, an adenovirus, or any combination thereof. The extracellular vesicle of claim 11, wherein the biologically active molecule is an inhibitor of a negative checkpoint regulator or an inhibitor of a binding partner of a negative checkpoint regulator. The extracellular vesicle of claim 21, characterized in that the negative checkpoint regulator is selected from the group consisting of: cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin mucin-containing protein 3 (TIM-3), B and T lymphocyte attenuation factor (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig inhibitor of T cell activation (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39 and CD73. The extracellular vesicle of claim 18, wherein the toxin is diphtheria toxin. The extracellular vesicle of claim 18, wherein the toxoid is tetanus toxoid. The extracellular vesicle of claim 18, wherein the non-toxic mutant of the toxin is a non-toxic mutant of diphtheria toxin. The extracellular vesicle of claim 18, wherein the positive co-stimulatory molecule is a member of the TNF receptor superfamily. 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. The extracellular vesicle of claim 18, wherein the activator of the positive co-stimulatory molecule is a member of the TNF superfamily. 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. The extracellular vesicle of claim 18, wherein the positive co-stimulatory molecule is a CD28 superfamily co-stimulatory molecule. The extracellular vesicle of claim 30, wherein the CD28 superfamily co-stimulatory molecule is ICOS or CD28. The extracellular vesicle of claim 18, wherein the activator of the positive co-stimulatory molecule is ICOSL, CD80 or CD86. 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. The extracellular vesicle of claim 19, characterized in that 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, programmed 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. The extracellular vesicle of claim 19, wherein the protein within the CRIPSR family includes Cas9 or Cas13. The extracellular vesicle of claim 18, wherein the polynucleotide comprises a DNA or RNA aptamer. The extracellular vesicle of any one of claims 1 to 36, wherein the extracellular vesicle is an exosome, a small vesicle or a large vesicle. A pharmaceutical composition comprising the extracellular vesicles according to any one of claims 1 to 37 and a pharmaceutically acceptable carrier. A cell that produces the extracellular vesicle of any one of claims 1-37. The cell according to claim 39, characterized in that it comprises a vector comprising a nucleic acid sequence encoding the scaffold protein and/or the biologically active molecule according to any one of claims 1 to 37. The cell of claim 40, wherein the nucleic acid sequence is operably linked to a promoter. A method for anchoring a bioactive molecule to an extracellular vesicle, comprising connecting the bioactive molecule to the scaffold protein according to any one of claims 1 to 37.