System and method for screening for gene affecting uptake of extracellular vesicle

Abstract

The present invention relates to a system and method for screening for a gene affecting the uptake of an extracellular vesicle. The screening system comprises: an extracellular vesicle-targeting region represented by formula A-B, a screening region represented by formula C-D-E, a first cell for integrating the extracellular vesicle-targeting region and / or the screening region, and a second cell for uptake of an extracellular vesicle, wherein A is a polypeptide localized to the extracellular vesicle, or a functional variant thereof, or a nucleotide sequence encoding the polypeptide or the functional variant, B is an RNA-binding polypeptide, or a functional variant thereof, or a nucleotide sequence encoding the polypeptide or the functional variant, C is a nucleotide sequence encoding a first RNA motif, D is a barcode sequence, E is a nucleotide sequence encoding a second RNA motif, the first RNA motif is complementary to a target sequence, and the second RNA motif is an RNA sequence specifically recognized and bound by B.

Description

Systems and methods for screening genes that influence the uptake of extracellular vesicles Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to systems and methods for screening genes that affect the uptake of extracellular vesicles, and their applications. Background Technology

[0002] Almost all mammalian cells secrete exosomes, which are nanoscale extracellular vesicles secreted by cells, with an average diameter of 30–200 nm. Exosomes are formed through a series of metabolic processes following continuous invagination of the cell membrane, resulting in a diverse composition. Encased in a lipid bilayer, exosomes carry various biomolecules, including proteins, glycans, lipids, and metabolites. When exosomes are absorbed by other cells, these substances are transferred and influence the phenotype of the recipient cell. Therefore, exosomes are considered an important medium for cell-cell communication, transmitting information to numerous cells and locations; the behavior of neighboring or distant cells can be altered by exosomes. Furthermore, donor cells can re-take up their own secreted exosomes. They play a crucial role in both physiological and pathological processes in the body.

[0003] Compared to synthetic carriers such as liposomes and nanoparticles, exosomes possess broad and unique advantages in disease diagnosis and treatment due to their endogenous and heterogeneous nature. However, issues such as storage stability, low yield, low purity, and weak targeting limit their clinical application. Furthermore, cost and technical challenges make it difficult to isolate large quantities of pure and specific exosomes from mixtures of different vesicle types in large solutions. Therefore, further research is needed to optimize these aspects and advance functional studies of exosomes. Summary of the Invention

[0004] To address one of the aforementioned technical problems in the prior art, this disclosure provides a high-throughput screening system that can screen for genes that can affect cellular uptake of extracellular vesicles, thereby enabling the large-scale purification of extracellular vesicles and providing new insights into the mechanism of cellular uptake of extracellular vesicles.

[0005] According to one aspect of this disclosure, a screening system is provided, the screening system comprising: (1) an external vesicle targeting region represented by formula AB, wherein A is a polypeptide or a functional variant thereof located in an external vesicle, or a nucleotide sequence thereof encoding the same, and B is an RNA-binding polypeptide or a functional variant thereof, or a nucleotide sequence thereof encoding the same; (2) a screening region represented by formula CDE, wherein C is a nucleotide sequence encoding a first RNA motif, D is a barcode sequence, and E is a nucleotide sequence encoding a second RNA motif, the first RNA motif being complementary to a target sequence, and the second RNA motif being an RNA sequence specifically recognized and bound by B; (3) a first cell for integrating the external vesicle targeting region and / or the screening region; and (4) a second cell for taking up the external vesicle, the external vesicle being secreted by a cell integrating the external vesicle targeting region and / or the screening region.

[0006] In some embodiments, the exovesicles include one or more of exosomes, microvesicles, apoptotic bodies, tumor vesicles, and nanovesicles.

[0007] In some embodiments, the polypeptide located on the outer vesicle is not particularly limited and theoretically includes any polypeptide located on the outer vesicle. In some embodiments, the polypeptide located on the outer vesicle includes, but is not limited to, CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71 (also known as transferrin receptor), CD133, CD138 (syndecan-1), CD235a, prostaglandin F2 receptor negative regulator (PTGFRN), brain acid-soluble protein 1 (BASP1), basigin (BSG) protein, protein kinase C substrate rich in myristyl alanine (MARCKS), protein kinase C substrate-like protein 1 rich in myristyl alanine (MARCKSL1), ALIX, and syndecan binding protein 1.SDCBP), Syntenin-2, Lamp2a, Lamp2b, TSPAN8, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, Integrin α-4 (ITGA4), Integrin β-1 (ITGB1), ITGB 5. ITGB6, ITGB7, 4F2 cell surface antigen heavy chain (SLC3A2), CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptor, interleukin receptor, immunoglobulin superfamily members (e.g., IGSF2, IGSF3, IGSF8), MHC-I or MHC-II components, CD2, CD3ε, CD3ζ, CD13, CD18, CD19, CD30, CD34, CD36, CD40, C D40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, C OL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, ARRDC1, LFA-1, LGALS3BP, Mac-1α, Mac-1β, MFGE8 PTGFRN, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, TSG101, VTI1A, VTI1B, fibronectin, RAB7A (also known as RAB7), 14-3-3ζ / δ, 14-3-3ε, HSC70, HSP90, HSPA13, ATP transporters (e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B, etc.), and any combination thereof, functional variants, fragments, or domains thereof. In some embodiments, the functional variants of the polypeptide located in the outer vesicles include those whose amino acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide located in the outer vesicles through the deletion, insertion, or substitution of one or more amino acids in its amino acid sequence.And it can still be located in the outer vesicle of a polypeptide.

[0008] In some embodiments, the polypeptide located on the outer vesicle may be a transmembrane or membrane-associated polypeptide. In some embodiments, the polypeptide located on the outer vesicle may be a polypeptide located inside the outer vesicle.

[0009] In some embodiments, the polypeptide located on the outer vesicle is selected from IGSF8, CD9, CD63, CD81, PTGFRN, BASP1, BSG, MARCKS, MARCKSL1, ALIX, or SDCBP, or any combination, functional variant, fragment, or domain thereof. In some embodiments, the polypeptide located on the outer vesicle can be BASP1, CD63, SDCBP, or any combination, functional variant, fragment, or domain thereof.

[0010] In some embodiments, the polypeptide BASP1 located in the outer vesicle may have an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0011] In some embodiments, the polypeptide CD63 located in the outer vesicle may have an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0012] In some embodiments, the polypeptide SDCBP located in the outer vesicle may have an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:3.

[0013] In some embodiments, the polypeptide or a functional variant of the outer vesicle forms a fusion protein with the RNA-binding polypeptide or a functional variant thereof. In some embodiments, the polypeptide or a functional variant of the outer vesicle is linked to the RNA-binding polypeptide or a functional variant thereof directly or via a linker.

[0014] In some embodiments, the RNA-binding polypeptide may be selected from, but is not limited to, Ku protein, Sm7 protein, MS2 capsid protein (MCP protein), λN protein, PP7 capsid protein (PCP protein), Com RNA-binding protein or aptamer, or any combination, functional variant, fragment, or domain thereof. In some embodiments, the RNA-binding polypeptide may be MS2 capsid protein (MCP protein), PP7 capsid protein (PCP protein), λN22 protein, or any combination, functional variant, fragment, or domain thereof.

[0015] In some embodiments, the RNA-binding polypeptide MCP protein may have an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:7.

[0016] In some embodiments, the RNA-binding polypeptide PCP protein may have an amino acid sequence as shown in SEQ ID NO:22, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:22.

[0017] In some embodiments, the RNA-binding polypeptide λN22 protein may have an amino acid sequence as shown in SEQ ID NO:23, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:23.

[0018] In some embodiments, the polypeptide located in the outer vesicle can be linked to the RNA-binding polypeptide via a linker.

[0019] In some embodiments, the fusion protein of this disclosure enables the RNA-binding polypeptide to be localized within the outer vesicle. In some embodiments, the RNA-binding polypeptide may be localized within the outer vesicle along with a therapeutic nucleic acid sequence.

[0020] In some embodiments, the second RNA motif may include one or more RNA sequences that are specifically recognized and bound by B. In some embodiments, the second RNA motif may be one or more selected from telomerase Ku-binding motifs, telomerase Sm7-binding motifs, MS2 phage operon stem loops, PP7 phage operon stem loops, boxB RNA stem loops, SfMu phage Com stem loops, non-natural RNA aptamers, etc.

[0021] In some embodiments, the RNA-binding polypeptide is capable of specifically binding to the second RNA motif. For example, RNA-binding polypeptide-RNA motif binding pairs may include Ku protein-Ku binding motif, Sm7 protein-telomerase Sm7 binding motif, MS2 capsid protein (MCP)-MS2 phage operon stem loop, PCP-PP7 phage operon stem loop, λN22 protein-boxB RNA stem loop, Com RNA-binding protein-Com stem loop, and aptamer ligand-corresponding RNA aptamer.

[0022] In some embodiments, the telomerase Ku binding motif may have a sequence such as 5'-UUCUUGUCGUACUUAUAGAUCGCUACGUUAUUUCAAUUUUGAAAAUCUGAGUCCUGGGAGUGCG-3' (SEQ ID NO:38).

[0023] In some embodiments, the telomerase Sm7 binding motif may have a sequence such as 5'-AAUUUUUGGA-3' (SEQ ID NO:39).

[0024] In some embodiments, the MS2 phage operon stem loop may have a sequence as shown in 5'-GCGCACAUGAGGAUCACCCAUGUGC-3' (SEQ ID NO:40).

[0025] In some embodiments, the PP7 phage operon stem loop may have a sequence as shown in 5'-GGAGCAGACGAUAUGGCGUCGCUCC-3' (SEQ ID NO:41).

[0026] In some embodiments, the SfMu phage Com stem loop may have a sequence such as 5'-CUGAAUGCCUGCGAGCAUC-3' (SEQ ID NO:42).

[0027] In some embodiments, the boxB RNA stem loop may have a sequence such as 5'-GGGCCCUGAAGAAGGGCCC-3' (SEQ ID NO:43).

[0028] In some embodiments, the second RNA motif may include one or more copies of a telomerase Ku-binding motif, a telomerase Sm7-binding motif, an MS2 phage operon stem loop, a PP7 phage operon stem loop, an SfMu phage Com stem loop, a non-natural RNA aptamer, etc. In some embodiments, the second RNA motif may include multiple copies of these RNA motifs.

[0029] In some embodiments, the first RNA motif may include, but is not limited to, one or more guide RNAs (sgRNAs), shRNAs, or siRNAs. The first RNA motif is complementary to one or more target sequences, knocking up, knocking down, or knocking out the expression of the one or more target sequences.

[0030] In some embodiments, the first RNA motif may be sgRNA. In this case, the system further includes a CRISPR-related protein coding region. In some embodiments, the CRISPR-related protein may be a CRISPR effector protein conventionally used in the art, capable of targeting specific gene (DNA) sequences under the guidance of sgRNA. In some embodiments, the CRISPR effector protein may be capable of targeting and activating or targeting and repressing the expression of specific genes.

[0031] In some embodiments, the CRISPR effector protein may be selected from Cas9, Cas12 (e.g., Cas12a, Cas12b) or Cas13 (e.g., Cas13a1, Cas13a2, Cas13b), or fragments thereof.

[0032] In some embodiments, the CRISPR effector protein may be Cas9, or a fragment thereof. In some embodiments, the CRISPR effector protein may have an amino acid sequence as shown in SEQ ID NO:11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:11.

[0033] In some embodiments, the outer vesicle targeting region may further include a CRISPR-associated protein coding region. In some embodiments, the outer vesicle targeting region, the RNA-binding polypeptide, or a functional variant thereof is linked to the CRISPR-associated protein (Cas) coding region via a linker coding region. In some embodiments, the linker may be selected from (GmS)n, (G)n, (EAAAK)n, or (XP)n, wherein m and n are each independently selected from integers from 1 to 5.

[0034] In some embodiments, the barcode sequence may indicate the sequence of the first RNA motif. In some embodiments, the sequence of the first RNA motif can be determined by sequencing the barcode sequence. In some embodiments, the barcode sequence may be a unique molecular identifier (UMI) sequence. There is a one-to-one correspondence between the barcode sequence and the first RNA motif; there may be one or more first RNA motifs and barcode sequences, with multiple first RNA motifs and their expected corresponding barcode sequences each corresponding to different genes.

[0035] In some embodiments, the outer vesicle targeting region is operatively connected to a first regulatory element. In some embodiments, the first regulatory element may be a type II promoter. In some embodiments, the first regulatory element may be selected from mammalian constitutive promoters, including, but not limited to, β-actin promoter (ACTB), CMV (cytomegalovirus) promoter; EF1A (elongation factor-1α) promoter; EFS promoter; CAG (composed of cytomegalovirus enhancer and chicken β-actin promoter); CBh promoter; SFFV promoter; MSCV promoter; SV40 (simian virus-derived) promoter; mPGK promoter; hPGK (phosphoglycerate kinase) promoter; UBC (ubiquitin C) promoter.

[0036] In some embodiments, the barcode sequence and the coding nucleotide sequence of the second RNA motif are tandemly transcribed in the screening region. In some embodiments, the coding nucleotide sequence of the first RNA motif is operatively linked to a second regulatory element, and the barcode sequence and the coding nucleotide sequence of the second RNA motif are operatively linked to a third regulatory element. In some embodiments, the second regulatory element and / or the third regulatory element can be a type III promoter, such as the U6 and H1 promoters. In some embodiments, the second regulatory element and the third regulatory element can be the same or different.

[0037] In some embodiments, the outer vesicle targeting region and the screening region may be located in the same carrier or in different carriers.

[0038] In some embodiments, the vector may be a non-viral vector or a viral vector. In some embodiments, the viral vector may be selected from adenovirus vectors, adeno-associated virus vectors (AAV), retroviral vectors, lentiviral vectors, Newcastle disease virus (NDV) vectors, lymphocytic choriomeningitis virus (LCMV) vectors, etc.

[0039] In some embodiments, the first cell can be any eukaryotic cell, such as any natural or modified eukaryotic cell. In some embodiments, the first cell is a mammalian cell. In some embodiments, the first cell includes, but is not limited to, CHO cells, HEK cells, Vero cells, mouse embryonic fibroblasts, hamster kidney fibroblasts, human liver cancer cells, or African green monkey kidney cells. In some specific embodiments, the first cell is a HEK 293T cell.

[0040] In some embodiments, the second cell can be any prokaryotic and / or eukaryotic cell, such as any natural or modified prokaryotic and / or eukaryotic cell. In some embodiments, the second cell can be an in vitro cell or an in vivo cell.

[0041] In some implementations, the first cell and the second cell are the same type or different types of cells.

[0042] In some embodiments, the second cell is selected from cells obtained by integrating the first cell with the outer vesicle targeting region and / or the screening region.

[0043] In some embodiments, the first cell and / or the second cell carries a selection tag. In some embodiments, the selection tag is selected from resistance genes, such as antibiotic resistance genes.

[0044] According to another aspect of this disclosure, a screening method is provided that uses the screening system to screen for genes that affect the uptake of extracellular vesicles.

[0045] In some implementations, the screening method includes the following steps:

[0046] (1) The extracellular vesicle target region and the screening region are transferred into the first cell to generate extracellular vesicles carrying barcode sequences;

[0047] (2) Co-culture the extracellular vesicles with the second cell;

[0048] (3) Isolate the RNA of the second cell and obtain the amount of the barcode sequence to determine the amount of the second cell taking in the external vesicles.

[0049] In some embodiments, step (3) includes obtaining the relative abundance (relative quantity) of the barcode sequence. If the relative abundance of the barcode sequence increases or decreases, it is determined that the gene corresponding to the barcode sequence information increases or decreases the uptake of external vesicles by the second cell. In some embodiments, the gene corresponding to the barcode sequence information is a gene in the first cell. In some embodiments, the uptake of external vesicles secreted by the first cell by the second cell is affected by knocking out or knocking down the target gene in the first cell using a first RNA motif.

[0050] In some embodiments, in step (2), after co-culturing extracellular vesicles carrying equal amounts of different barcode sequences with the second cell, the function of the gene corresponding to the barcode sequence information affecting extracellular vesicle uptake can be determined by directly comparing the content of each barcode sequence in the second cell.

[0051] In some embodiments, the method further includes detecting the relative abundance of different barcode sequences in the external vesicles obtained in step (1) prior to step (2). In some embodiments, in step (3), the relative abundance of the barcode sequences can be calculated using methods known to those skilled in the art.

[0052] In a specific implementation, the relative abundance of the barcode sequence can be calculated from the relative abundance of different barcode sequences in the second cell and the relative abundance of different barcode sequences in the external vesicles obtained in step (1). For example, the rate of change of the barcode sequence (UMI) can be obtained by the following formula: UMI rate of change = (relative abundance of UMI in the second cell / relative abundance of UMI in the external vesicles - 1) * 100%), where the UMI information in the second cell is obtained by cDNA reverse transcription of the RNA of the second cell; the relative abundance of different UMIs in the second cell is the ratio of the amount of each UMI to the total amount of UMIs; the relative abundance of different UMIs in the external vesicles is the ratio of the amount of each UMI in the external vesicles to the total amount of UMIs.

[0053] In some implementations, the target sequence is extrachromosomal DNA and / or genomic DNA.

[0054] In some specific embodiments, the screening method includes:

[0055] (1) The outer vesicle target region and the selection region are transferred into the first cell to obtain cells expressing the outer vesicle target region and the selection region;

[0056] (2) Separate extracellular vesicles produced by the cells obtained in step (1), wherein the extracellular vesicles carry barcode sequences;

[0057] (3) Co-culture the extracellular vesicles with the second cell;

[0058] (4) Analyze the barcode sequences in the co-cultured cells obtained in step (3) to screen for genes that affect the amount of extracellular vesicles taken up.

[0059] In some implementations, step (4) includes: extracting total RNA from co-cultured cells, reverse transcribing it to obtain cDNA, and analyzing the barcode sequence information contained therein to determine the function of the gene corresponding to the barcode sequence information.

[0060] According to another aspect of this disclosure, the use of the screening system or the screening method in the preparation of medicaments for diagnosing or treating diseases using extracellular vesicles is provided.

[0061] According to another aspect of this disclosure, the use of the screening system or the screening method in screening for genes that affect extravesicle uptake is provided.

[0062] According to another aspect of this disclosure, the use of the screening system or the screening method in cells that secrete targeted extracellular vesicles is provided.

[0063] This disclosure utilizes a highly efficient and intuitive high-throughput screening system to rapidly and efficiently screen for genes related to the uptake of extracellular vesicles, eliminating the need for individual gene verification. Genes are labeled with tags, and the corresponding gene information is obtained by verifying the tags. The larger the number of genes to be screened, the more obvious the advantages of this approach become. Furthermore, it features a short research cycle and is intuitive and reliable. Attached Figure Description

[0064] Figure 1 illustrates, exemplarily, the flow of a gene screening method for influencing extracellular vesicle production according to one embodiment of the present disclosure.

[0065] Figure 2 illustrates, exemplarily, the structure of a lentivirus expression system according to one embodiment of the present disclosure.

[0066] Figure 3 illustrates an sgRNA vector structure according to one embodiment of the present disclosure.

[0067] Figure 4 illustrates an example of an expression plasmid for an external vesicle cytoskeleton protein.

[0068] Figure 5 shows the morphology and particle size distribution of exosomes according to one embodiment of this disclosure. Figure A shows a photograph of the morphology of cell-generated exosomes as detected by transmission electron microscopy; Figure B shows the particle size distribution of exosomes as detected by Nanoflow.

[0069] Figure 6 shows the protein distribution and expression results on exosomes according to one embodiment of this disclosure. A represents the protein distribution on exosomes; B represents the expression of fluorescent proteins in exosomes.

[0070] Figure 7 exemplarily illustrates expression plasmids for the exovesicle cytoskeleton protein and sgRNA according to one embodiment of the present disclosure. A is a schematic diagram of the expression plasmid for the exovesicle cytoskeleton protein and the RNA-binding polypeptide; B is a schematic diagram of the expression plasmid for sgRNA.

[0071] Figure 8 shows the morphology and particle size distribution of exosomes according to one embodiment of this disclosure. Figure A shows a photograph of the morphology of cell-generated exosomes as detected by transmission electron microscopy; Figure B shows the particle size distribution of exosomes as detected by Nanoflow.

[0072] Figure 9 shows the relative expression level of UMI in exosomes according to one embodiment of the present disclosure.

[0073] Figure 10 shows the morphology and particle size distribution of exosomes according to one embodiment of the present disclosure. A-C show the morphology (top) and particle size distribution (bottom) of exosomes secreted by 293T cells transfected with MCP / MS2, PCP / PP7, and λN22 / boxB, respectively.

[0074] Figure 11 shows the relative expression level of UMI in exosomes according to one embodiment of the present disclosure.

[0075] Figure 12 shows the expression result of BASP1 according to one embodiment of the present disclosure.

[0076] Figure 13 shows the results of sgRNA transfection with RAB7 or CD276 according to one embodiment of this disclosure. In Figure 13, A and C show the protein expression results and their quantification results after sgRNA transfection with RAB7, respectively; B and D show the protein expression results and their quantification results after sgRNA transfection with CD276, respectively.

[0077] Figure 14 shows the morphology and particle size distribution of exosomes according to one embodiment of the present disclosure.

[0078] Figure 15 shows the results of detecting the expression of various proteins in exosomes according to one embodiment of the present disclosure, wherein the second lane is a cell sample of 293T cells and the other lanes are exosome samples.

[0079] Figure 16 shows the results of detecting UMI expression in exosomes according to one embodiment of this disclosure. The figure shows the results after normalizing the amount of UMI relative to the amount of GAPDH.

[0080] Figure 17 shows the electron microscopic observation results of exosomes obtained according to one embodiment of the present disclosure.

[0081] Figure 18 shows the results of exosome abundance comparison of an embodiment of this disclosure.

[0082] Figure 19 shows the results of exosome abundance comparison in one embodiment of this disclosure.

[0083] Figure 20 shows a comparison of the uptake of exosomes produced by cells under the influence of different genes in one embodiment of this disclosure. Detailed Implementation

[0084] This invention relates to a gene screening method that affects the uptake of extracellular vesicles. Based on a high-throughput gene screening system, the method analyzes the uptake of extracellular vesicles produced under the influence of different genes and screens out genes that can affect the ability of extracellular vesicles to be taken up.

[0085] This invention relates to a gene screening method that affects the uptake of extracellular vesicles. Based on a high-throughput gene screening system, the method analyzes the uptake of extracellular vesicles produced under the influence of different genes and screens out genes that can affect the ability of extracellular vesicles to be taken up.

[0086] The screening system of the present invention comprises: (1) an external vesicle targeting region represented by formula AB, wherein A is a polypeptide or a functional variant thereof located in an external vesicle, or a nucleotide sequence thereof encoding the polypeptide or the target polypeptide or a functional variant thereof encoding the target polypeptide or ...

[0087] The first RNA motif acts as a gene intervention factor, intervening in genes within the cell. The barcode sequence linked to the first RNA motif specifically binds to extracellular vesicles, which serve as localization structures. The intervened first cell produces extracellular vesicles carrying the barcode sequence. These barcode-carrying extracellular vesicles are then co-cultured with a second cell. Analysis of the barcode sequences within the co-cultured cells allows for the screening of genes corresponding to the number of barcode sequences; these genes are those influencing the uptake of extracellular vesicles. This invention's method is characterized by its short cycle time, intuitiveness, and reliability.

[0088] In the screening region, there can be one or more first RNA motifs, each corresponding to a different gene. After exerting their effects in different cells, their gene functions are studied. One or more first RNA motifs can knock up, knock out, or knock down a gene in the cell screening environment. Barcode sequences corresponding one-to-one with the first RNA motifs are designed, or barcode sequences can specifically affect the first RNA motifs, thereby establishing a one-to-one correspondence between barcode sequences and first RNA motifs. The barcode sequences can connect to the extracellular vesicle target region. Extracellular vesicles may be affected by the intervention of the first RNA motif on specific genes, specifically by affecting their quantity, location, cellular uptake localization structure, or cellular uptake. After a series of targeted treatments, by collecting extracellular vesicles or extracellular vesicle vectors linked to RNA-binding proteins, and combining the quantity information, location information, and one or more of the linked barcode sequence information of the extracellular vesicles, the functional information of the gene corresponding to the first RNA motif is determined.

[0089] The screening environment is a cellular environment, and the extracellular vesicle target region and the screening region can appear in the same cellular screening environment. The extracellular vesicle carrier linked to the RNA-binding protein is a cell or organelle containing extracellular vesicles linked to the RNA-binding protein. The RNA-binding protein is linked to a specific protein on the extracellular vesicle, which is an extracellular vesicle membrane protein or an extracellular vesicle wall protein. When it is an extracellular vesicle membrane protein, it must be a transmembrane protein with an extracellular segment. The specific protein can be one or more of BASP1, MARCKSL1, MARCKS, BSG, IGSF8, and PTGFRN. The RNA-binding protein can be one or more of MCP, λN, and PCP. The structure on the barcode sequence used for identification can be a molecular tag UMI, and it has a second RNA motif that can be linked to the RNA-binding protein. Depending on the type of RNA-binding protein, it can be one or more of MS2, BoxB, and PP7. When using the RNA-binding protein and the second RNA motif, the pairing is MCP-MS2, λN-BoxB, or PCP-PP7. When a barcode sequence and an extracellular vesicle linked to an RNA-binding protein are in the same cellular environment, they can be linked through the interaction between the RNA-binding protein and the second RNA motif, thus linking the extracellular vesicle with the corresponding barcode sequence information.

[0090] During implementation, plasmids that express the target region of extracellular vesicles and plasmids that express the selection region invade the same cell selection environment, generating extracellular vesicles linked with barcode sequences. These extracellular vesicles linked with barcode sequences can be taken up by a second cell. By counting the number of tag structures taken up by the second cell, it is determined which tag has the most elements, and the corresponding gene can be inferred to affect the ability of extracellular vesicles to be taken up.

[0091] The first RNA motif can intervene in genes through enhancement, knockdown, or deletion. In some embodiments, sgRNA can be used as the first RNA motif, and a gene can be knocked out by Cas9 protein under the action of sgRNA, thereby affecting the uptake of extracellular vesicles. When using this technique to intervene in genes, sgRNAs that are one-to-one matched with the genes to be screened are constructed on the barcode sequence. In other embodiments, the enhanced expression of a specific gene can also be achieved; for example, a Cas9 that has lost its endonuclease activity can be used, and a transcriptional enhancer can be linked to the Cas9. Under the guidance of sgRNA, it can target and bind to a position within 200 bp upstream of the transcription start site, enhancing the expression of the gene. The enhanced expression of a gene will also affect extracellular vesicles, which act as localization molecules. In still other embodiments, gene knockdown technology can also be used to intervene in genes in the screened cells. siRNA or shRNA can be designed and transferred into the screened cells to achieve a knockdown effect by inhibiting the gene. Whether it is enhancement, knockdown, or deletion, these are all methods of gene interference and can all be used in this scheme. Accordingly, the first RNA motif can be sgRNA, shRNA, or siRNA. The first RNA motif itself or in conjunction with gene intervention tools can achieve gene intervention in the screening cell environment.

[0092] In some embodiments, this disclosure utilizes the CRISPR / Cas system. In a specific embodiment, this disclosure utilizes the CRISPR / Cas9 system. Cas9 is a type II CRISPR (clustered regularly spaced short palindromic repeats) system derived from microorganisms, which has been shown to cleave DNA when paired with a single guide RNA (gRNA). Cas9 primarily cleaves genomic sites where the gRNA sequence is followed by a PAM sequence (-NGG).

[0093] In some implementations, CRISPR / Cas system nucleases require sgRNA to cleave genomic DNA. These sgRNAs consist of: (1) a 19–21 nucleotide spacer of a variable sequence (guide sequence) that enables the CRISPR / Cas system nuclease to target genomic sites in a sequence-specific manner, and (2) an invariant hairpin sequence that is constant between the guide RNA and allows the guide RNA to bind to the CRISPR / Cas system nuclease. In the presence of the CRISPR / Cas nuclease, the guide RNA triggers a CRISPR / Cas-based genome cleavage event in the cell. It should be understood that any hairpin sequence can be used, as long as it can be recognized and bound by the CRISPR / Cas nuclease.

[0094] In some embodiments, the system of this disclosure may further include a coding sequence for a reporter factor. In some embodiments, the reporter factor is used to identify potentially transfected cells and to assess the function of regulatory sequences. Generally, the reporter factor is not an endogenous or native gene of the host cell, and the protein it encodes can be readily determined. In some embodiments, the reporter factor is known in the art and includes, but is not limited to, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cell surface markers, resistance genes (such as neo antibiotics), etc.

[0095] Figure 1 illustrates, exemplarily, a method for screening genes that affect the uptake of extracellular vesicles according to a specific embodiment of the present disclosure, which may include the following steps.

[0096] Step 1: Construct plasmids expressing sgRNA and UMI-linked sequences (the structure may be as shown in Figure 2 in some embodiments of this invention), and package them using lentivirus. Corresponding sgRNAs and UMIs can be designed according to the selected genes, with each set of sgRNAs and UMIs corresponding to one gene. In some embodiments, an sgRNA library can be used, and a corresponding UMI can be designed for each sgRNA, thereby enabling the screening of unknown genes.

[0097] Step 2: Construct plasmids capable of expressing the specific protein-RNA binding protein and Cas9 (the structure in some embodiments of this invention may be as shown in Figure 3) and package them using lentiviruses. The coding sequences for the Cas9 protein and the specific protein-RNA binding protein can be constructed on the same plasmid to simultaneously infect the first cell, or they can be constructed on different plasmids to sequentially infect the first cell.

[0098] Step 3: First, lentiviruses containing plasmids expressing specific proteins—RNA-binding proteins and Cas9—are used to infect the first cell line to obtain monoclonal cells. Then, multiple lentiviruses containing plasmids expressing sgRNA and UMI-linking sequences are used to infect the aforementioned monoclonal cells. There is no particular restriction on the ratio of lentiviruses to cells, but preferably no higher than 0.5:1, aiming to ensure that only one plasmid is introduced into each cell, intervening in only one gene. After culturing, extracellular vesicles are extracted; tag structures are attached to the extracellular vesicles. Total RNA is extracted from a portion of the extracellular vesicles, and the UMI content is detected using RT-qPCR.

[0099] Step 4: Co-culture some of the extracellular vesicles obtained in Step 3 with monoclonal cells, wash away extracellular vesicles that could not be taken up into the cells, extract total RNA from the cells, and detect the UMI content using RT-qPCR; sort according to the amount of tag information to screen for genes that affect the uptake of extracellular vesicles.

[0100] Using the above method, genes that affect the uptake of extracellular vesicles can be quickly screened from multiple genes to be screened. Furthermore, based on the difference in UMI content, the functional differences between two different genes can be preliminarily determined.

[0101] Those skilled in the art will understand that, following step (2), a gRNA-UMI expression construct targeting multiple target genes can be constructed, or a gRNA-UMI library can be constructed. High-throughput sequencing can then be used to detect the abundance of each UMI in exosomes produced by the first cell after infection with the lentiviral library and the abundance of each UMI taken up by the second cell, in order to screen for genes affecting extracellular vesicle or exosome uptake. In some embodiments, the genes affecting extracellular vesicle or exosome uptake may include genes that increase or decrease exosome uptake.

[0102] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0103] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0104] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0105] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0106] As used herein, the terms “extracellular vesicle,” “extracellular vesicle,” or “EV” refer to cell-derived vesicles containing a membrane encapsulating an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, microvesicles, apoptotic bodies, tumor vesicles, nanovesicles, etc.) with a diameter smaller than that of the cell from which they originate. In some aspects, extracellular vesicles have a diameter ranging from 20 nm to 1000 nm and may contain various macromolecular payloads within an internal space (i.e., a lumen), displayed on the outer surface of the extracellular vesicle, and / or transmembrane. In some aspects, said payloads may include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some aspects, the extracellular medium includes a scaffold portion. By way of example, and not limitation, extracellular vesicles include apoptotic bodies, cell debris, cell-derived vesicles obtained by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicular organelles, and vesicles generated by living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). Extracellular vesicles can originate from living or dead organisms, explant tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some respects, extracellular vesicles are produced by cells expressing one or more transgenic products.

[0107] As used herein, the term "exosome" refers to an extracellular vesicle with a diameter between 30 and 200 nm (e.g., between 40 and 200 nm). Exosomes comprise a membrane enclosing their internal space and, in some aspects, can be produced by cells via direct plasma membrane budding or via the fusion of late endosomes or multivesicles with the plasma membrane. In some 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 combinations thereof. In some aspects, the EVs (e.g., exosomes) of this disclosure are produced by cells expressing one or more transgenic products. In some embodiments, the diameter of the exosomes can be 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm.

[0108] As used herein, the terms “membrane-associated protein” or “membrane-associated polypeptide” refer to proteins or peptides that are associated with the membrane of the exovesicle, and may include proteins integrated into the membrane of the exovesicle, protruding outside or inside the exovesicle.

[0109] As used herein, the term "substitution" or "replacement" of amino acids can refer to the substitution of a conserved amino acid residue, wherein the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such substitution is considered conserved. In another respect, a string of amino acids can be conservatively replaced by a structurally similar string that differs in the order and / or composition of its side chain family members.

[0110] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0111] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (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 percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0112] As used herein, the term "host cell" refers to the cell used to produce external vesicles. Host cells can be cells cultured in vitro or cells in vivo. Host cells include, but are not limited to, cells known to be effective at producing external vesicles, such as HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), fibroblasts, and neuronal precursor cells (e.g., AGE.HN). ○R Neuronal precursor cells), amniotic fluid cells (e.g., CAP) ○R Amniotic fluid cells, adipose-derived mesenchymal stem cells, and RPTEC / TERT1 cells.

[0113] As used herein, the terms “individual,” “subject,” “host,” and “patient” are used interchangeably to refer to any mammalian subject, particularly a human, in the presence of a diagnostic, therapeutic, or therapeutic agent. The compositions and methods described herein are suitable for therapeutic and veterinary applications in humans. In some respects, the subject is a mammal; in others, the subject is a human. As used herein, “mammal subject” includes all mammals, including but not limited to humans, domesticated animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0114] As used herein, the term "linker" refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. The domains in the bispecific fusion polypeptide of the present invention can be linked by linkers, wherein each linker is fused to and / or otherwise linked (e.g., via peptide bonds) with at least two polypeptides or domains. In some embodiments, all linkers present in the bispecific fusion polypeptide of the present invention have the same amino acid sequence. In other embodiments, at least two linkers present in the bispecific fusion polypeptide of the present invention have different amino acid sequences. The linker should have a length suitable for linking two or more monomeric domains in this manner, and the linker should ensure that the different domains to which it is linked fold correctly and are properly presented to perform their biologically active functions. In various embodiments, the linker has a flexible conformation. Suitable flexible linkers include, for example, those having glycine, glutamine, and / or serine residues. In some embodiments, the linker may be selected from (Glycine, glutamine, and / or serine residues). n S) m (G) n (EAAAK) n Or (XP) n , where n and m are each independently selected from integers from 0 to 5. For example, n is selected from 0, 1, 2, 3, 4, or 5, and m is selected from 1, 2, 3, 4, or 5. In some embodiments, the connector may also include, for example, KESGSVSSEQLAQFRSLD (SEQ ID NO:44), EGKSSGSGSESKST (SEQ ID NO:45), GGGGGGGG (SEQ ID NO:46), GSAGSAAGSGEF (SEQ ID NO:47), etc.

[0115] As used herein, the term "barcode sequence" refers to a barcode chain portion containing a nucleic acid sequence representing spatial, sequencing information, and / or encoded data. The barcode sequence can be pre-determined by a barcode library. The barcode sequence can be a sequence containing DNA, RNA, synthetic nucleosides, or any combination thereof. In some embodiments of this disclosure, the barcode sequence can be unique and correspond to a linked sgRNA. During sequencing, the barcode sequence can be used to distinguish samples or as a nucleotide sequence segment to distinguish different test sequences within a sample. As those skilled in the art will understand, the length of the barcode sequence can be determined as needed and based on library capacity. Those skilled in the art will also understand that barcode sequences can be used to distinguish different sequencing samples in commonly used next-generation sequencing platforms (such as the Illumina sequencing platform). In some embodiments, the length of the barcode sequence can be approximately 6–30 bp, for example, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, or 30 bp. In some embodiments, the barcode sequence may include a unique molecular identifier (UMI).

[0116] As used herein, the term "operably linked" refers to the connection of at least a first element and a second element such that the constituent elements are in a relationship that allows them to function in their intended manner. For example, a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence if a regulatory sequence (e.g., a promoter sequence) and a coding sequence are linked in a manner that allows the expression of the coding sequence to be controlled by the regulatory sequence. In some embodiments, the "operably linked" regulatory sequence is covalently bound to the coding sequence, directly or indirectly (e.g., in a single nucleic acid molecule). In some embodiments, the regulatory sequence controls the expression of the coding sequence in a trans-regulatory manner, and including the regulatory sequence in the same nucleic acid as the coding sequence is not a requirement for operational linking.

[0117] Recombinant expression vectors may contain the nucleic acids described in this invention, in a form suitable for expression in host cells. This means that the recombinant expression vector includes one or more regulatory elements, selectable according to the host cell for expression, which are operatively linked to the nucleic acid sequence to be expressed. Within the recombinant expression vector, "operative linking" is intended to link the target nucleotide sequence to the regulatory element in a manner that allows for nucleotide expression (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced).

[0118] Type III promoters (U6, H1) primarily express non-coding short RNA sequences, such as sgRNA, shRNA, and miRNA.

[0119] Type II promoters can be further divided into three main categories based on their usage characteristics: constitutive promoters (widespread promoters), tissue-specific promoters, and inducible promoters. Specific promoters can only be expressed in specific cells, making them suitable for animal experiments. Inducible promoters require the addition of specific drugs to activate or inhibit their activity, such as tetracycline-induced TRE / TRE3G expression. In expression systems, constitutive promoters (widespread promoters) are the most widely used. These promoters maintain a certain level of expression activity in most cells; however, different promoters may exhibit varying levels of expression.

[0120] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection 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.

[0121] Example

[0122] Example 1: Testing the effect of different peptides on the localization of external vesicles.

[0123] The encoding nucleic acid sequences of the extravesicular skeletal proteins BASP1, CD63, and SDCBP were constructed into the pcDNA3.1(+) plasmid (universal biological) vector, respectively, to obtain plasmids pcDNA3.1-BASP1-EGFP-Nanoluc, pcDNA3.1-CD63-EGFP-Nanoluc, and pcDNA3.1-SDCBP-EGFP-Nanoluc. For ease of detection, EGFP-Nanoluc was linked to the C-terminus of BASP1, CD63, and SDCBP. In simple terms, according to the amino acid sequences given in Table 1 and those conventionally used in the art, the nucleic acid sequences encoding BASP1, CD63, SDCBP, EGFP, and Nanoluc were synthesized and inserted into the multiple cloning site of the pcDNA3.1(+) vector to obtain the target plasmids. Figure 4 illustrates the constructed plasmids.

[0124] Table 1

[0125] 20,000 pieces / cm 2293T cells were seeded at a density of [insert density here] in T225 cell culture flasks and cultured overnight in DMEM (Gibco) medium containing 10% fetal bovine serum. 293T cells were transfected with plasmids pcDNA3.1-BASP1-EGFP-Nanoluc, pcDNA3.1-CD63-EGFP-Nanoluc, and pcDNA3.1-SDCBP-EGFP-Nanoluc, respectively. Transfection with pcDNA3.1 plasmid served as a control. After 72 hours, the cell culture supernatant was collected, and exosomes were isolated and purified.

[0126] The morphology and particle size distribution of exosomes in different groups were detected by transmission electron microscopy and Nanoflow. The results are shown in Figure 5. The transmission electron microscopy images show that the exosomes produced by the cells in each group have a typical saucer-shaped morphology (A in Figure 5). The Nanoflow detection results show that the exosome particle size is mainly distributed between 30 and 200 nm (B in Figure 5).

[0127] Furthermore, the proportion of exosomes containing green fluorescence in each group was detected using the FITC channel of Nanoflow, thus revealing the distribution ratio of the BASP1 / CD63 / SDCBP proteins fused with these proteins in the exosomes. Nano-glo was used to detect the expression level of Nanoluc in each group of exosomes (a stronger luminescence signal indicates a higher Nanoluc expression level), thereby revealing the content of the fused BASP1 / CD63 / SDCBP proteins in the exosomes. The results are shown in Figure 6. Figure 6A shows that BASP1 is more widely distributed in exosomes than CD63 and SDCBP proteins. Figure 6B shows that BASP1 has a higher content in exosomes than CD63 and SDCBP proteins.

[0128] Example 2: Comparison of the loading effects of different peptides located in the outer vesicles on UMI molecules.

[0129] As shown in Figure 7A, plasmids Lenti-CMV-BASP1-MCP-EF-1α-Cas9, Lenti-CMV-CD63-MCP-EF-1α-Cas9, and Lenti-CMV-SDCBP-MCP-EF-1α-Cas9 were constructed (with a FLAG tag between BASP1 and MCP). In short, following the amino acid sequences given in Tables 1 and 2 and those conventionally used in the field, the nucleic acid sequences of the CMV promoter, BASP1, CD63, SDCBP, and MCP were synthesized and inserted between the cPPT / CTS and EF-1αcore promoter elements of plasmid LentiCas9-Blast (Addgene, #52962).

[0130] As shown in Figure 7B, construct the Lenti-mU6-sgRNA-hU6-NC-MS2 plasmid. In simple terms, following the sequence given in Table 2, synthesize the mU6-sgRNA-hU6-NC-MS2-CMV promoter-EGFP-P2A sequence. First, remove the U6-gRNA scaffold fragment from the LentiGuide-Puro plasmid (Addgene, #52963). Then, replace the EF-1α promoter fragment on the plasmid with the synthesized mU6-sgRNA-hU6-NC-MS2-CMV promoter-EGFP-P2A fragment to obtain the target plasmid.

[0131] Table 2.

[0132] At 50,000 cells / cm 2 293T cells were seeded at a density of [insert density here] in T225 cell culture flasks and cultured overnight in DMEM (Gibco) medium containing 10% fetal bovine serum. Plasmids 1 and 2 were co-transfected into the cells according to Table 3 below. Transfection with LentiCas9-Blast plasmid was used as a control. After 72 hours, the cell culture supernatant was collected, and exosomes were isolated and purified.

[0133] Table 3.

[0134] The morphology (Figure 8A) and particle size distribution (Figure 8B) of exosomes from different groups were examined using transmission electron microscopy and Nanoflow.

[0135] In addition, using the miRNeasy Serum / Plasma Kit (50) (Qiagen), 200 μL of exosomes from each group were extracted for total RNA extraction according to the instructions. Equal amounts of RNA were then used to perform primer pairing.

[0136] UMI-F: 5'-TAACAACTCCGCTCCAAGGC-3' (SEQ ID NO: 20);

[0137] UMI-R: 5'-GGTGATCCTCATGTTTCGATGAAG-3' (SEQ ID NO:21) was subjected to RT-qPCR to analyze the loading of UMI molecules in exosomes of each group. The results are shown in Figure 9.

[0138] Example 3: Comparison of the loading effects of different RNA-binding proteins / RNAs on UMI in exosomes

[0139] In Example 2, the MCP in the plasmid Lenti-CMV-BASP1-MCP-EF-1α-Cas9 was replaced with PCP or λN22, resulting in plasmids Lenti-BASP1-PCP and Lenti-BASP1-λN22, respectively. Correspondingly, MS2 in the sgRNA-NC-MS2 plasmid was replaced with PP7 or boxB. Table 4 below shows the amino acid sequences of PCP and λN22, as well as the coding nucleic acid sequences of PP7 and boxB.

[0140] Table 4.

[0141] 20,000 pieces / cm 2 At a density of [insert density here], 293T cells were seeded in T225 cell culture flasks and cultured overnight in DMEM medium (Gibco) containing 10% fetal bovine serum. The cells were then co-transfected with plasmid 1 and plasmid 2 according to Table 5 below.

[0142] After 72 hours, the cell culture supernatant was collected, and the exosomes were isolated and purified.

[0143] Table 5.

[0144] The morphology (Figure 10, A-C, top) and particle size distribution (Figure 10, A-C, bottom) of different groups of exosomes were detected using transmission electron microscopy and Nanoflow.

[0145] In addition, according to the instructions, 200 μL of exosomes from each group were taken for total RNA extraction using the miRNeasy Serum / Plasma Kit (50) (Qiagen). An equal amount of RNA was then used to extract RNA using primer pairs.

[0146] UMI-F: 5'-TAACAACTCCGCTCCAAGGC-3' (SEQ ID NO: 20);

[0147] UMI-R: 5'-GGTGATCCTCATGTTTCGATGAAG-3' (SEQ ID NO:21) was used for RT-qPCR to analyze the loading of UMI molecules in exosomes of each group. The results are shown in Figure 11. The results show that when using different RNAs and their corresponding RNA-binding peptides, MCP / MS2 has a significantly better loading effect on UMI molecules than PCP / PP7 and λN22 / boxB.

[0148] Example 4: Construction of BASP1-MCP monoclonal cells

[0149] In this embodiment, the Lenti-CMV-BASP1-MCP-EF-1α-Cas9 plasmid constructed in Example 2 was used and packaged into a lentivirus by an outsourced service company (Genewiz).

[0150] To validate the system disclosed herein, corresponding sgRNA and UMI sequences were designed based on the RAB7 and CD276 gene sequence information (as shown in Table 6 below). Following the pattern shown in Figure 7B, the sgRNA-UMI-MS2 plasmid was constructed and named RAB7 sgRNA-UMI-MS2 and CD276 sgRNA-UMI-MS2, respectively. The NC sgRNA-UMI-MS2 plasmid was used as a control plasmid.

[0151] Table 6.

[0152] HEK293 T cells were seeded in 6-well plates at a density of 2 × 10⁶ cells / well. 5 Cells / well, cultured overnight at 37°C. HEK293 T cells were infected with Lenti-CMV-BASP1-MCP-EF-1α-Cas9 lentivirus. Wells not infected with lentivirus were used as control wells. After 24 hours, the medium was replaced with DMEM complete medium containing 10% fetal bovine serum (BSD final concentration 20 μg / mL). The medium was changed every 3 days until all cells in the control wells died.

[0153] Cells from the experimental group were digested with trypsin, diluted, and seeded into 96-well plates. 300 μL of DMEM complete medium containing BSD and 10% fetal bovine serum was added to each well, resulting in approximately 70% empty wells. The plates were incubated at 37°C. Cells in the 96-well plates were observed every two days. Once sufficient cell proliferation was achieved in the wells containing monoclonal cells, the cells were digested with trypsin, seeded into 24-well plates, and cultured further to gradually scale up the cell culture system, obtaining BASP1-MCP monoclonal cells BASP-MCP-BSD-293T-1 and BASP-MCP-BSD-293T-2.

[0154] Western blotting was used to detect the expression of BASP-1 (flag-tagged) in cell lines BASP-MCP-BSD-293T-1 and BASP-MCP-BSD-293T-2, as well as in uninfected lentiviral control cells 293T-1 and 293T-2. Representative results are shown in Figure 12. The BASP1-MCP monoclonal cell line with high expression levels (BASP-MCP-BSD-293T-2, or simply 293T-2) was selected for subsequent experiments.

[0155] Example 5. Infection of BASP1-MCP monoclonal cells using the sgRNA-UMI-MS2 expression vector.

[0156] The BASP1-MCP monoclonal cells (293T-2) selected in Example 4 were seeded into 6-well plates, 2 × 10⁻⁶. 5 Cells / well, cultured overnight at 37°C. BASP1-MCP monoclonal cells and 293T cells were infected with RAB7 sgRNA-UMI-MS2, CD276 sgRNA-UMI-MS2, or NC sgRNA-UMI-MS2 lentiviruses, respectively. The lentivirus-to-cell ratio was controlled at 0.3–0.5:1. Uninfected BASP1-MCP monoclonal cells and 293T cells were used as controls. After 24 hours, the cells were replaced with DMEM complete medium containing Puro and 10% fetal bovine serum (Puro final concentration 10 μg / mL). The medium was changed every 3 days until all cells in the control group died.

[0157] Western blotting was used to detect the expression of RAB7, CD276, BASP-1 (flag-tagged), and Cas9 proteins in cells using primary antibodies against RAB7 (Abcam, ab137029), CD276 (Abcam, ab134161), anti-flag (Abcam, ab125243), and anti-Cas9 (Abcam, ab189380), respectively. The results are shown in Figures 13A-D. As can be seen from Figures 13A-D, infection with RAB7 sgRNA-UMI-MS2 and CD276 sgRNA-UMI-MS2 significantly reduced the expression of the target genes RAB7 and CD276.

[0158] Cells infected with different lentiviruses were seeded into T225 cell culture flasks at a density of 20,000 cells / cm³. 2 After 72 hours, the cell culture supernatant was collected, and exosomes were isolated and purified. The morphology and particle size distribution of exosomes in different groups were detected by transmission electron microscopy and Nanoflow microscopy, respectively. The results are shown in Figure 14.

[0159] The first row of Figure 14 shows the morphology of exosomes produced by different groups of cells, which resembles a cup-shaped structure and is typical of exosomes. The second row shows the particle size distribution of exosomes produced by different groups of cells, which ranges from 30 to 200 nm.

[0160] In addition, the protein expression on the isolated exosomes was examined. In short, Western blotting was used to detect the expression of CD81, TSG101, GM130, and Calexin proteins in exosomes using primary antibodies against anti-CD81 (Abcam, ab109201), anti-TSG101 (Abcam, ab125011), anti-GM130 (Abcam, ab52649), and anti-Calexin (Abcam, ab112995). The results are shown in Figure 15. Figure 15 shows that CD81 and TSG101, the two exosome surface markers, were detected in exosomes, while the exosome-negative proteins GM130 and Calexin were not detected, as expected. In 293T cells, GM130 and Calexin proteins were detected, while CD81 and TSG101 were almost not expressed.

[0161] In addition, the relative content of gene-specific UMIs in exosomes was detected. In short, total RNA was extracted from exosomes using the Qiagen miRNeasy Serum / Plasma Kit (Qiagen 217184), and reverse transcription (20 μL system) was performed using the All-in-One First-Strand Synthesis MasterMix (with dsDNase) (Yugong Life, EG15133S) reagent according to the reagent instructions. 4 μL of the reverse transcription product was diluted 10-fold, and 2 μL was added to a 20 μL qPCR system. TB... Premix Ex Taq TM The UMI content in exosomes was detected by qPCR using the II (Tli RNaseH Plus) (TaKaRa RR820B) reagent, following the instructions.

[0162] The qPCR primers are as follows:

[0163] UMI-F: 5'-TAACAACTCCGCTCCAAGGC-3' (SEQ ID NO: 20);

[0164] UMI-R: 5'-GGTGATCCTCATGTTTCGATGAAG-3' (SEQ ID NO: 21);

[0165] GAPDH-F: 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID NO: 30);

[0166] GAPDH-R: 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID NO: 31).

[0167] Figure 16 shows the results of qPCR detection, indicating the relative content of gene-specific UMIs carried in exosomes, demonstrating that the exosomal scaffold protein fused with MCP can facilitate the entry of gene-specific UMIs into exosomes. The gRNA results are from 293 cells (uninfected with BASP1-MCP lentivirus) infected with RAB7 sgRNA-UMI-MS2, CD276 sgRNA-UMI-MS2, and NC sgRNA-UMI-MS2 lentiviruses, respectively.

[0168] Example 6: Study on the Influence of Genes on Exosome Uptake

[0169] The effects of RAB7A, CD276, and PD-L1 genes on exosome uptake were analyzed. Following the procedures described in Example 2, gRNA-UMI plasmids containing these three genes were constructed: RAB7A-sgRNA-UMI-MS2, CD276-sgRNA-UMI-MS2, PD-L1-sgRNA-UMI-MS2, and the control plasmid NC-sgRNA-UMI-MS2. Based on publicly available sequence information for genes RAB7A (NCBI Gene ID: 7879), CD276 (NCBI Gene ID: 80381), and PD-L1 (NCBI Gene ID: 29126), corresponding sgRNA and UMI sequences were designed. The sgRNA and UMI sequence information for different genes is shown in Table 7. Other components for plasmid construction can use existing commonly used components or be directly custom-made commercially. The constructed plasmids were then packaged into lentiviruses.

[0170] Table 7.

[0171] The BASP1-MCP monoclonal cells obtained in Example 4 were seeded into 6-well plates, 2 × 10⁶ cells per well. 5Cells / well, cultured overnight at 37°C. RAB7A-sgRNA-UMI-MS2, CD276-sgRNA-UMI-MS2, PD-L1-sgRNA-UMI-MS2, and NC-sgRNA-UMI-MS2 lentiviruses were mixed at the same titer and used to infect BASP1-MCP monoclonal cells, maintaining a lentivirus to cell ratio of 0.3-0.5:1. Cells in wells not infected with lentivirus served as controls. After 24 hours, the cells were replaced with DMEM complete medium containing puromycin and 10% fetal bovine serum (Puro final concentration 10 μg / ml). The medium was changed every 3 days until all cells in the control group died, yielding a stable mixed cell line expressing BASP1-MCP-gRNA-UMI-MS2.

[0172] Exosome extraction: Stable transfected BASP1-MCP-gRNA-UMI-MS2 cells obtained through screening were seeded in 1L shake flasks at a density of 5×10⁶ cells / year. 5 Cells / mL, cultured in 300mL OPM-293CD05 Medium (Shanghai OPM), in suspension culture. After 72h, the cell culture supernatant was collected, and 500μL of exosomes were obtained by separation and purification using ultrafiltration and size exclusion methods. The purified exosomes were characterized, and the results are shown in Figure 17. Total RNA was extracted from exosomes using Qiagen's miRNeasy Kit (50). 100ng of RNA was used to perform reverse transcription (20μL system) using All-in-One First-Strand Synthesis MasterMix (with dsDNase) (Yugong Life, EG15133S) reagent according to the reagent instructions. 4μL of reverse transcription product was taken, diluted 10-fold, and 2μL was added to 20μL of qPCR system. TB Premix Ex Taq TM The abundance values ​​of different UMI species in exosomes were determined using reagent II (Tli RNaseH Plus) (TaKaRa RR820B) (primer sequences are shown in Table 8 below). The results are shown in Figure 18.

[0173] Table 8.

[0174] Analysis of the uptake of different exosomes: 2 × 10⁶ exosomes were seeded in each well of a 6-well plate. 5293S cells were cultured overnight in 2 mL of DMEM medium (containing 10% FBS). 100 μL of exosomes (containing 4 UMIs) from the stable mixed cells obtained in the previous step were added to the 293S cells. After co-incubation for 6 hours, the medium was aspirated, and the cells were washed three times with PBS. The cells in the container were then digested with trypsin for 3 min, and the trypsin was neutralized with complete medium. The cell suspension was centrifuged at 300g for 5 min, and resuspended and washed twice with PBS. Finally, the centrifuged cells were collected and processed using Qiagen's... Total RNA was extracted from cells using the Mini Kit (50). 100 ng of RNA was used to perform reverse transcription (20 μL system) using the All-in-One First-Strand Synthesis MasterMix (with dsDNase) reagent (Yugong Life, EG15133S) according to the reagent instructions. 4 μL of the reverse transcription product was diluted 10-fold, and 2 μL was added to a 20 μL qPCR system. TB... Premix Ex Taq TM Using reagent II (Tli RNaseH Plus) (TaKaRa RR820B) and primers listed in Table 8, the UMI content was detected, and the results are shown in Figure 19. Based on the results in Figures 18 and 19, the rate of change of UMI was calculated, as shown in Figure 20 (Formula: UMI rate of change = (relative abundance of UMI in the second cell / relative abundance of UMI in the extravesicles - 1) * 100%). As shown in Figure 20, exosomes produced under the influence of CD276 and PD-L1 genes are easily taken up by 293S cells, while exosomes produced under the influence of RAB7 gene are not easily taken up by 293S cells.

[0175] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A screening system, characterized in that, The screening system includes: (1) The target region of the external vesicle shown in formula AB, where A is a polypeptide or its functional variant located in the external vesicle, or its encoded nucleotide sequence, and B is an RNA-binding polypeptide or its functional variant, or its encoded nucleotide sequence. (2) The screening region shown in formula CDE, where C is the nucleotide sequence encoding the first RNA motif, D is the barcode sequence, and E is the nucleotide sequence encoding the second RNA motif. The first RNA motif is complementary to the target sequence, and the second RNA motif is an RNA sequence that is specifically recognized and bound by B. (3) A first cell, for integrating the outer vesicle targeting region and / or the screening region; and (4) A second cell for taking in external vesicles, which are secreted by cells that integrate the external vesicle target region and / or the screening region.

2. The screening system according to claim 1, characterized in that, The exovesicles include one or more of exosomes, microvesicles, apoptotic bodies, tumor vesicles, and nanovesicles; and / or The polypeptide located in the outer vesicle is a transmembrane or membrane-associated polypeptide, or a polypeptide located inside the outer vesicle. Preferably, the polypeptide located in the outer vesicle is selected from IGSF8, CD9, CD63, CD81, PTGFRN, BASP1, BSG, MARCKS, MARCKSL1, ALIX, or SDCBP, more preferably from BASP1, CD63, or SDCBP, or any combination thereof, functional variant, fragment, or domain thereof, and / or The RNA-binding polypeptide is selected from Ku protein, Sm7 protein, MS2 capsid protein, λN protein, PP7 capsid protein, Com RNA-binding protein or aptamer ligand, more preferably from MS2 capsid protein, PP7 capsid protein, λN22 protein, or any combination, functional variant, fragment or domain thereof.

3. The screening system according to claim 1, characterized in that, The second RNA motif is selected from one or more of the following: telomerase Ku-binding motif, telomerase Sm7-binding motif, MS2 phage operon stem-loop, PP7 phage operon stem-loop, SfMu phage Com stem-loop, boxB RNA stem-loop, and non-natural RNA aptamers; more preferably, it is selected from MS2 phage operon stem-loop, PP7 phage stem-loop, or boxB RNA stem-loop; and / or The first RNA motif is selected from one or more guide RNAs (sgRNA), shRNA, or siRNA; Preferably, the expression of the target sequence is knocked up, knocked down, or knocked out by the first RNA motif.

4. The screening system according to claim 1, characterized in that, The first RNA motif is one or more sgRNAs. Preferably, the outer vesicle targeting region further includes a CRISPR effector protein coding region, preferably selected from Cas9, Cas12 or Cas13, or fragments thereof.

5. The screening system according to claim 1, characterized in that, The external vesicle targeting region is operably connected to a first regulatory element, preferably a type II promoter, more preferably a mammalian constitutive promoter; and / or The barcode sequence and the encoding nucleotide sequence of the second RNA motif are transcribed in tandem. Preferably, the coding nucleotide sequence of the first RNA motif is operatively linked to a second regulatory element. Preferably, the barcode sequence and the coding nucleotide sequence of the second RNA motif are operatively linked to a third regulatory element. More preferably, the second control element and / or the third control element are type III promoters, and more preferably, each is an independent U6 or H1 promoter.

6. The screening system according to claim 1, characterized in that, The outer vesicle targeting region and the screening region may be located in the same carrier or in different carriers. Preferably, the vector is a viral vector, more preferably selected from adenovirus vectors, adeno-associated virus vectors (AAV), retroviral vectors, lentiviral vectors, Newcastle disease virus (NDV) vectors, or lymphocyte choriomeningitis virus (LCMV) vectors.

7. The screening system according to claim 1, characterized in that, The first cell is a eukaryotic cell, preferably a mammalian cell, more preferably selected from one or more of CHO cells, HEK cells, Vero cells, mouse embryonic fibroblasts, hamster kidney fibroblasts, human liver cancer cells, and African green monkey kidney cells; and / or The second cell is a prokaryotic cell and / or a eukaryotic cell; and / or The first cell and / or the second cell carries a selection tag, preferably selected from a resistance gene, more preferably an antibiotic resistance gene.

8. A screening method, characterized in that, The screening method uses the screening system according to any one of claims 1 to 7 to screen for genes that affect the uptake of extracellular vesicles.

9. The method according to claim 8, characterized in that, The screening method includes: (1) The extracellular vesicle target region and the screening region are transferred into the first cell to generate extracellular vesicles carrying barcode sequences; (2) Co-culture the extracellular vesicles with the second cell; (3) Isolate the RNA of the second cell to obtain the amount of the barcode sequence in order to determine the amount of the second cell taking in external vesicles; Preferably, the method further includes detecting the abundance of extracellular vesicles carrying different barcode sequences before step (2).

10. Use of the screening system according to any one of claims 1 to 7 or the screening method according to claim 8 or 9 in the preparation of a medicament for diagnosing or treating a disease, preferably, the diagnosis or treatment is performed using extracellular vesicles.

11. Use of the screening system according to any one of claims 1 to 7 or the screening method according to claim 8 or 9 in screening for genes affecting extracellular vesicle uptake or in preparing cells that secrete targeted extracellular vesicles.

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