Fate recording system for extracellular vesicles

Abstract

The present invention provides a fate recording system for extracellular vesicles. The system is realized by inserting and recording, in a receiver cell of an extracellular vesicle, a specific sequence included in the extracellular vesicle.

Description

Extracellular vesicle fate recording system

[0001] <Cross Reference> This application claims priority from U.S. Provisional Patent Application No. 63 / 485,570, filed February 17, 2023, the entire contents of which are incorporated herein by reference. All publications and patents cited throughout the body of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) are incorporated by reference in their entirety.

[0002] The present invention relates to a system for recording the fate of extracellular vesicles.

[0003] It has been revealed that there are several types of vesicles (extracellular vesicles; EVs) secreted from cells, depending on their origin and characteristics. Numerous classification methods exist based on their heterogeneity, but from the perspective of size, they can be roughly divided into EVs with a diameter of 200 nm or less (small EVs) and larger EVs. The former are membrane vesicles with a diameter of approximately 30-200 nm, and contain many exosomes, whose membranes are thought to be primarily derived from endosomes. While exosomes are heterogeneous, many are believed to be rich in endosome-associated proteins such as Rab GTPase, SNARE, annexin, and phloritin, as well as the transmembrane protein family tetraspanins (CD63, CD81, CD9, etc.). Large EVs include microvesicles (MVs; also known as ectosomes), whose main components are thought to be cell membrane components, and apoptotic bodies formed by cell fragmentation during apoptosis (Non-Patent Documents 1, 2, and 3). MVs are formed by being directly constricted from the cell membrane and are small vesicles approximately 200 nm to 1000 nm in size, containing integrins, selectins, CD40, and the like. Apoptotic bodies are also formed by being directly constricted from the cell membrane, but are small vesicles 500 nm to 2000 nm in size, encapsulating fragmented genomic DNA, histone proteins, and the like.

[0004] Exosomes have been reported to be involved in close and long-distance intercellular communication. For example, in the immune system, exosomes released from cells function as antigen-presenting vesicles, inducing antitumor immune responses and immune tolerance that suppresses inflammation (Non-Patent Document 4). In neurodegenerative diseases, pathogenic proteins such as prions and beta-amyloid peptides are known to use exosomes to propagate to other cells.

[0005] Exosomes have low immunogenicity and the ability to penetrate the blood-brain barrier. They contain various nucleic acids (e.g., mRNA, miRNA, shRNA, and ncRNA) in addition to proteins, and these nucleic acids are known to function within exosome-receiving cells and affect their function. Therefore, in recent years, active research has been conducted into the use of exosomes as drug delivery systems (DDS) in hopes of achieving therapeutic effects by modifying the function of exosome-receiving cells (Non-Patent Document 5). For example, attempts have been made to improve the efficiency of DDS by expressing peptides or proteins recognized by exosome-receiving cells on the exosome membrane surface to increase the efficiency of targeting to exosome-receiving cells, or by expressing RNA-binding proteins on the exosome inner membrane to efficiently recruit cytoplasmic RNA (Non-Patent Documents 6-9, Patent Document 1).

[0006] The present inventors have also developed exosomes suitable for DDS (Non-Patent Document 10).

[0007] It has also been proposed that cancer cells program their metastatic destinations using exosomes they secrete, creating an environment favorable for their own metastasis. Therefore, some papers suggest that if exosome secretion could be selectively inhibited in cancer cells, anticancer drugs could be developed (Non-Patent Documents 11 and 12). These studies suggest that the type of integrin present on the surface of exosomes is related to the type of organ to which cancer metastasizes.

[0008] Extracellular vesicles are not only unique to animals but also exist in plants, and it has been suggested that they play an important role in the host's defense against pathogens (plant immunity), but the function of extracellular vesicles is not well understood (Non-Patent Document 13).

[0009] The present inventors have succeeded in creating a library of extracellular vesicles encapsulating nucleic acids for comprehensive screening of factors that affect the properties of extracellular vesicles (Patent Document 2).

[0010] US Publication No. 2015 / 0093433 International Publication No. 2021 / 095842 Special Publication No. 2023-525304 US Publication No. 2023 / 0383289 US Publication No. 2023 / 0357766 International Publication No. 2020 / 053239

[0011] eLife (2018); 7: e41460 Journal of Extracellular Vesicles (2015); 4: 26316 Journal of Extracellular Vesicles (2018); 7:1535750 Immunological Reviews (2013); Vol. 251:p125-142NATURE (2017);VOL546:p498-521Nano letters (2019);19:19-28Journal of Extracellular Vesicles (2016); 5:31027Journal of Extracellular Vesicles (2016);5:31053Nature Biotechnology (2011);29:341-345NATURE COMMUNICATIONS (2018); 9:1305NATURE (2015); VOL527 (7578): p329-35Sci. Rep. (2018); 8:8161 Plant Physiology (2017); Vol. 173:p728-741NATURE COMMUNICATIONS (2017);8:15178Nature. 2019 December; 576 (7785): p149-157Molecular Threapy. 29, (2021): p1729-1743. Cell. 2014 October 23;159(3):p635-646Dev Cell. 2020 December 21; 55(6): p784-801. e9Nature Biotechnology volume 39, (2021): p198-206 Genome Biology (2021) 22:235

[0012] The present invention aims to provide improved barcoded extracellular vesicles, donor cells for extracellular vesicles, receptor cells for extracellular vesicles, methods for producing them, and methods for using them.

[0013] As a result of extensive research, the present inventors have succeeded in incorporating a fusion protein containing a PrimeEditing gRNA containing a sequence complementary to the barcode sequence and a PrimeEditor enzyme containing a nuclear localization signal into extracellular vesicles. When a receptor cell that has taken up the extracellular vesicles contains a nucleic acid encoding a target sequence for the PrimeEditing gRNA in its nucleus, the barcode sequence is inserted into the target sequence by a genome editing reaction.

[0014] The present invention includes the following embodiments: (Extracellular Vesicles) [A1] Extracellular vesicles comprising: (a) a fusion protein comprising a PrimeEditor enzyme comprising a nuclear localization signal; and (b) a PrimeEditing gRNA comprising a complementary strand sequence of a barcode sequence. [A2] The extracellular vesicles according to [A1], comprising: (a) a fusion protein comprising a PrimeEditor enzyme comprising a first Tag peptide and a nuclear localization signal; (b) a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence; and (c) a fusion protein of a Tag peptide-binding protein present in the extracellular vesicles, the Tag peptide-binding protein binding to a second Tag peptide and the first Tag peptide, wherein the binding affinity of the second Tag peptide is greater than the binding affinity of the first Tag peptide. [A3] The extracellular vesicle according to [A2], wherein the first tag peptide comprises AlfaTagPE (SEQ ID NO: 21), the second tag peptide comprises AlfaTagST (SEQ ID NO: 22), and the tag-binding protein comprises an anti-Alfa nanobody (SEQ ID NO: 27). [A4] The extracellular vesicle according to [A1], comprising: (a) a fusion protein comprising a first antibody and a PrimeEditor enzyme comprising a nuclear localization signal; (b) a PrimeEditing gRNA comprising a complementary strand sequence of a barcode sequence; and (c) a fusion protein of an antigenic peptide that binds to the first antibody and a protein present in the extracellular vesicle. [A5] The extracellular vesicle according to [A4], wherein the antigenic peptide is SUNtag peptide (GNC peptide x10) (SEQ ID NO: 24) and the first antibody is an anti-GCN single-chain antibody (SEQ ID NO: 25). [A6] The extracellular vesicle according to any one of [A1] to [A5], further comprising (d) a viral fusion protein on its surface. [A7] The extracellular vesicle according to [A6], wherein (d) the viral fusion protein comprises VSV-G. [A8] The extracellular vesicle according to any one of [A1] to [A7], wherein the protein present in the extracellular vesicle comprises CD9 (SEQ ID NO: 28) or BASP1 (1-30) (SEQ ID NO: 29).[A8] The extracellular vesicle according to any one of [A2] to [A8], wherein the protein present in the extracellular vesicle (c) further comprises a peptide corresponding to the barcode sequence and is presented outside the vesicle. [A9] The extracellular vesicle according to any one of [A1] to [A8], wherein the barcode sequence comprises any nucleic acid sequence of 5 to 8 bases. [A10] The extracellular vesicle according to [A9], wherein the barcode sequence is 5'NNNNNNTGNN3' (where N is any base). [A11] The extracellular vesicle according to [A9], wherein the PrimeEditing gRNA comprising a complementary strand sequence of the (b) barcode sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 13 to 20 and 63. [A12] The extracellular vesicles according to any one of [A1] to [A11], having an average diameter of 30 nm or more and 150 nm or less.

[0015] (Receiver cell) [B1] (i) A cell containing in its nucleus a nucleic acid encoding a target sequence for a PrimeEditing gRNA; preferably wherein, when the extracellular vesicle described in [A1] is taken up into the cell, a fusion protein containing a PrimeEditor enzyme containing an (a) nuclear localization signal, which encapsulates a PrimeEditing gRNA containing a complementary strand sequence of the (b) barcode sequence, is translocated into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction. [B2] A cell (i) containing a nucleic acid encoding a target sequence for the PrimeEditing gRNA in its nucleus; and (ii) containing a fusion protein comprising the second Tag peptide in its cytoplasm; preferably wherein, when the extracellular vesicles described in [A2] are taken up into the cell, the adhesion between the (c) Tag peptide-binding protein and the (a) fusion protein comprising the PrimeEditor enzyme comprising the first Tag peptide and a nuclear localization signal is dissociated due to the antagonism with the fusion protein comprising the second Tag peptide, resulting in the (b) fusion protein comprising the PrimeEditing gRNA containing the complementary strand sequence of the barcode sequence (a) first Tag peptide and the PrimeEditor enzyme comprising the nuclear localization signal being translocated into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction. [B3] The first Tag peptide is AlfaTag PE (SEQ ID NO: 21), and the second Tag peptide is AlfaTag ST(SEQ ID NO: 22), and the tag-binding protein comprises an anti-Alfa nanobody (SEQ ID NO: 27). [B4] (i) A cell comprising in its nucleus a nucleic acid encoding a target sequence for a PrimeEditing gRNA; preferably wherein, when the extracellular vesicle described in [A4] is taken up into the cell, the adhesion between the antigen peptide of (c) and the first antibody of (a) is dissociated, resulting in the translocation of a fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal (a) encapsulating a PrimeEditing gRNA containing a complementary strand sequence of the barcode sequence (b) into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction. [B5] The target sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 6 below, [B6] The cell according to any one of [B1] to [B4]. [B6] The cell according to any one of [B1] to [B5], which comprises a sequence encoding a fluorescent protein and / or a labeling protein, and wherein the fluorescent protein or labeling protein is transcribed and translated only when the barcode sequence is inserted into a target sequence. [B7] The cell according to any one of [B1] to [B6], which is selected from the group consisting of HEK293 cells, stem cells, epithelial cells, endothelial cells, fibroblasts, cancer cells, immune cells, nerve cells, and plant cells.

[0016] (Donor cell) [C1] A cell that secretes extracellular vesicles of [A1], comprising: (A) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal of (a); and (B) a nucleic acid encoding a PrimeEditing gRNA containing a complementary strand sequence of the barcode sequence of (b). [C2] A cell that secretes the extracellular vesicles of [A2], comprising: (A) a nucleic acid encoding a fusion protein comprising the first tag peptide of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) a nucleic acid encoding a fusion protein of a tag peptide-binding protein present in the extracellular vesicles and a protein that binds to the second tag peptide of (c) and the first tag peptide, wherein the binding affinity for the second tag peptide is greater than the binding affinity for the first tag peptide. [C3] A cell that secretes the extracellular vesicles of [A4], comprising: (A) a nucleic acid encoding a fusion protein comprising the first antibody of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) a nucleic acid encoding a fusion protein of an antigen peptide that binds to the first antibody of (c) and a protein present in the extracellular vesicles. [C4] The extracellular vesicle-secreting cell of any one of [C1] to [C3], further comprising a nucleic acid encoding the viral fusion protein of (D) (d). [C5] The extracellular vesicle-secreting cell of any one of [C1] to [C4], wherein the fusion protein with the protein present in the extracellular vesicles of (C) (c) contains a peptide corresponding to the barcode sequence and is presented outside the vesicles. [C6] The extracellular vesicle-secreting cell according to any one of [C1] to [C4], further comprising (E) a nucleic acid that affects the properties of extracellular vesicles, or an expression vector that expresses a nucleic acid that affects the properties of extracellular vesicles.[C7] The extracellular vesicle-secreting cell according to [C6], wherein the nucleic acid that affects the properties of the extracellular vesicles is selected from the group consisting of: (1) a nucleic acid that changes the amount of an endogenous protein present within or on the surface of the extracellular vesicles; (2) a nucleic acid that promotes or inhibits the secretion of extracellular vesicles; (3) a nucleic acid that affects the lipid membrane that constitutes the membrane of the extracellular vesicles; and (4) a nucleic acid that causes an exogenous protein to be present within or on the surface of the extracellular vesicles. [C8] The extracellular vesicle-secreting cell according to claim C6, wherein the nucleic acid that affects the properties of the extracellular vesicles comprises mRNA, ncRNA, crRNA, or gRNA. [C9] The extracellular vesicle-secreting cell according to [C8], further expressing Cas12a (Cpf1) endonuclease. [C10] The extracellular vesicle-secreting cell according to any one of [C1] to [C9], wherein the extracellular vesicle-secreting cell is selected from the group consisting of HEK293 cells, stem cells, epithelial cells, endothelial cells, fibroblasts, cancer cells, immune cells, nerve cells, and plant cells.

[0017] [D1] A non-human organism comprising or consisting of a cell according to any one of [B1] to [B7].

[0018] (Nucleic acid for producing donor cells) [E1] At least one nucleic acid selected from the following group for producing the cell of [C1]: (A) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal of (a); and (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b). [E2] At least one nucleic acid selected from the following group for producing the cell of [C2]: (A) a nucleic acid encoding a fusion protein comprising the first tag peptide of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) a nucleic acid encoding a fusion protein of a tag peptide-binding protein present in extracellular vesicles and a protein that binds to the second tag peptide of (c), wherein the binding affinity for the second tag peptide is greater than the binding affinity for the first tag peptide. [E3] At least one nucleic acid selected from the group consisting of (A) a nucleic acid encoding a fusion protein comprising the first antibody of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) a nucleic acid encoding a fusion protein of an antigen peptide that binds to the first antibody of (c) and a protein present in extracellular vesicles. [E4] A nucleic acid encoding (D) a viral fusion protein of (d) for producing the cell of [C4]. [E5] The nucleic acid of any one of [E1] to [E3], wherein the fusion protein with the protein present in extracellular vesicles of (C) (c) contains a peptide corresponding to the barcode sequence and is presented outside the vesicles. [E6] The nucleic acid of any one of [E1] to [E5], which is encapsulated in an expression vector. [E7] (E) A nucleic acid that affects the properties of extracellular vesicles, or an expression vector that expresses a nucleic acid that affects the properties of extracellular vesicles, for producing the cell of [C7].[E8] The nucleic acid or expression vector according to [E7], wherein the nucleic acid that affects the properties of extracellular vesicles is selected from the group consisting of: (1) a nucleic acid that changes the amount of an endogenous protein present within or on the surface of extracellular vesicles; (2) a nucleic acid that promotes or inhibits the secretion of extracellular vesicles; (3) a nucleic acid that affects the lipid membrane that constitutes the membrane of extracellular vesicles; and (4) a nucleic acid that causes an exogenous protein to be present within or on the surface of extracellular vesicles. [E9] The nucleic acid or expression vector according to [E7], wherein the nucleic acid that affects the properties of extracellular vesicles comprises mRNA or ncRNA (including miRNA, siRNA, shRNA, snRNA, snoRNA, gRNA, and sgRNA). [E10] An expression vector for expressing Cas12a (Cpf1) endonuclease to produce the cell of [C9].

[0019] (Nucleic acids for producing receiver cells) [F1] For producing the cell of [B1] or [B4], (i) a nucleic acid encoding a target sequence for a Prime Editing gRNA. [F2] For producing the cell of [B2], at least one nucleic acid selected from the following group: (i) a nucleic acid encoding a target sequence for a Prime Editing gRNA; and (ii) a nucleic acid encoding a fusion protein comprising a second Tag peptide. [F3] The nucleic acid of [F1] or [F2] further comprises a sequence encoding a fluorescent protein and / or a labeling protein, and is configured so that the fluorescent protein or labeling protein is transcribed and translated only when the barcode sequence is inserted into the target sequence. [F4] The nucleic acid of any one of [F1] to [F3], which is contained in an expression vector.

[0020] (Method for preparing a library) [G1] A method for preparing a library of extracellular vesicles, comprising: 1) introducing into an extracellular vesicle-secreting cell: (i) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme comprising a nuclear localization signal, or an expression vector for expressing the fusion protein; and (ii) a combination of multiple types of a1) a nucleic acid comprising a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence of at least one barcode sequence; and a2) a nucleic acid comprising at least one sequence that affects the properties of extracellular vesicles corresponding to the barcode sequence; or multiple types of a) a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence of at least one barcode sequence; and at least one sequence that affects the properties of extracellular vesicles corresponding to the barcode sequence; 2) culturing the extracellular vesicle-secreting cells after the introduction in a culture medium; and 3) A method comprising a step of recovering extracellular vesicles containing the PrimeEditor enzyme and the PrimeEditing gRNA from the culture supernatant of extracellular vesicle-secreting cells. [G2] The method according to [G1], wherein the extracellular vesicle-secreting cells express Cas12a (Cpf1) endonuclease, and the sequence that affects the properties of the extracellular vesicles is a gRNA or sgRNA sequence for an endogenous gene.[G3] A method for producing a library of extracellular vesicles that display peptides on their outer surface, comprising: 1) introducing into an extracellular vesicle-secreting cell: (i) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme comprising a nuclear localization signal, or an expression vector for expressing the fusion protein; and (ii) a combination of: a) a nucleic acid comprising a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence to at least one barcode sequence; and b) a nucleic acid comprising a sequence encoding a fusion protein comprising a peptide corresponding to the barcode sequence and a protein present in the extracellular vesicles that is to be presented outside the vesicles; or a combination of: a1) a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence to at least one barcode sequence; and a2) a sequence encoding a fusion protein comprising a peptide corresponding to the barcode sequence and a protein present in the extracellular vesicles that is to be presented outside the vesicles; 2) culturing the extracellular vesicle-secreting cells after the introduction in a culture medium; and 3) A method comprising the step of recovering extracellular vesicles that contain a fusion protein comprising the PrimeEditor enzyme and the PrimeEditing gRNA and present the peptide from the culture supernatant of extracellular vesicle-secreting cells. [G4] The method of any one of [G1] to [G3], comprising the step, prior to step 2), of introducing a nucleic acid comprising a sequence encoding a fusion protein of a tag peptide-binding protein and a protein present in extracellular vesicles, the tag peptide-binding protein being a protein that binds to a second tag peptide and the first tag peptide, wherein the binding affinity for the second tag peptide is greater than the binding affinity for the first tag peptide, wherein the fusion protein comprising the PrimeEditor enzyme and the nuclear localization signal of (i) contains the first tag peptide.[G5] The method of any one of [G1] to [G3], comprising, before step 2), a step of introducing a nucleic acid comprising a sequence encoding a fusion protein of a tag peptide-binding protein that binds to a second tag peptide and the first tag peptide, wherein the tag peptide-binding protein has a greater binding affinity for the second tag peptide than for the first tag peptide, and a protein present in extracellular vesicles, wherein the fusion protein comprising a PrimeEditor enzyme comprising a nuclear localization signal of (i) comprises the first tag peptide. [G6] The method of [G5], wherein the first tag peptide comprises AlfaTagPE (SEQ ID NO: 21), the second tag peptide comprises AlfaTagST (SEQ ID NO: 22), and the tag-binding protein comprises an anti-Alfa Nanobody (SEQ ID NO: 27). [G7] The method of any one of [G1] to [G3], further comprising, before step 2), a step of introducing a nucleic acid comprising a sequence encoding a fusion protein of an antigenic peptide that binds to a first antibody and a protein present in extracellular vesicles, wherein the fusion protein (i) comprising a PrimeEditor enzyme comprising a nuclear localization signal comprises the first antibody. [G8] The method of [G7], wherein the antigenic peptide is SUNtag peptide (GNC peptide x10) (SEQ ID NO: 24) and the first antibody is an anti-GCN antibody (SEQ ID NO: 25). [G9] The method of any one of [G1] to [G8], further comprising, before step 2), a step (d) of introducing a nucleic acid comprising a sequence encoding a viral fusion protein. [G10] The method of any one of [G1] to [G9], wherein the protein present in extracellular vesicles comprises CD9 (SEQ ID NO: 28) or BASP1 (1-30) (SEQ ID NO: 29). [G11] The method according to any one of [G1] to [G10], wherein the barcode sequence comprises any nucleic acid sequence of 5 to 8 bases. [G12] The method according to [G11], wherein the barcode sequence is 5'NNNNNNTGNN3' (wherein N is any base). [G13] The method according to any one of [G1] to [G12], wherein the PrimeEditing gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 13 to 20 and 63.

[0021] The present invention can be useful for the development of an efficient drug delivery system using extracellular vesicles, biology research on extracellular vesicles, and drug discovery research targeting the extracellular vesicle secretion pathway.

[0022] FIG. 1B shows the structure of a sequence containing a target sequence for receiver cells, which is one embodiment of the present invention. Insertion of a target sequence into the target sequence results in normal expression of GFP and luciferase. FIG. 1B shows the efficiency of prime editing using pegRNAs #1 to #6 corresponding to target sequences #1 to #6 in Example 1. FIG. 2A shows a schematic diagram of pegRNA sequences. FIG. 2B shows the efficiency of prime editing using different pegRNA sequences in Example 2. FIG. 3A shows a schematic diagram of extracellular vesicles, which is one embodiment of the present invention, and a schematic diagram of sequence information for constructing the structure. FIG. 3B shows the amount of pegRNA in extracellular vesicles, which is one embodiment of the present invention. FIG. 4A shows a schematic diagram of an extracellular vesicle fate recording system, which is one embodiment of the present invention, and a schematic diagram of sequence information for constructing the structure. FIG. 4B shows the editing efficiency in Example 4 (flow cytometry). FIG. 4C shows the editing efficiency in Example 4 (PCR: restriction enzyme). A) AGGAAT; B) AGGACA; C) CATTCA; D) GCCTAA; E) TGTCGA. Figure 5A is a schematic diagram of sequence information for creating a construct for inserting a longer barcode sequence into a target sequence, according to one embodiment of the present invention. Figure 5B shows the editing efficiency in Example 5 (luciferase assay).

[0023] The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustrative or explanatory purposes, and are not intended to limit the present invention thereto. It will be apparent to those skilled in the art that various changes and modifications can be made based on the description in this specification within the spirit and scope of the present invention disclosed herein. In this disclosure, the term "comprising" includes "essentially comprising" and "consisting of" aspects.

[0024] The present invention includes extracellular vesicles comprising: (a) a fusion protein comprising a PrimeEditor enzyme comprising a nuclear localization signal; and (b) a PrimeEditing gRNA comprising a complementary strand sequence of a barcode sequence.

[0025] Extracellular (secretory) vesicles (EVs) are vesicles used to release intracellular substances extracellularly and are formed by a phospholipid bilayer. Examples of lipid compositions include sphingomyelin and phosphatidylserine. Their size ranges from 10 nm to 10 μm, 30 nm to 5000 nm, or 50 nm to 3000 nm in diameter. Small EVs (mainly including exosomes and microvesicles) with diameters of 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, or 50 nm or more, and 500 nm or less, 400 nm or less, 300 nm, or 200 nm or less are preferred in the present invention, but are not limited thereto. Their origin is preferably derived from eukaryotes, but is not particularly limited thereto. Extracellular vesicles derived from humans, non-human mammals (including mice and rats), higher plants, and microorganisms (including enterobacteria) are preferred.

[0026] The PrimeEditor enzyme containing a nuclear localization signal is a fusion protein used in PrimeEditing that contains i) a nucleic acid-programmed DNA-binding protein (napDNAbp) and (ii) a polypeptide having RNA-dependent DNA polymerase activity (Patent Documents 3 to 5; Non-Patent Document 15). The napDNAbp preferably has nickase activity, and may be selected from the group consisting of Cas9 (including SpCas9, SaCas9, and mutants lacking endonuclease activity (dCas9)), Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, CasX, CasY, Argonaute, and active fragments thereof. In the case of Cas9 having a RuvC domain and an HNH domain, it is preferable that the activity of the HNH domain is deleted and that the Cas9 has the activity of introducing nicks into single strands (i.e., nickase activity). Although not particularly limited, Cas9 may have nickase activity by having an amino acid substitution of H840A. Such Cas9 may have the native amino acid sequence other than H840A, or may further include amino acid substitutions, deletions, and additions as long as its activity is maintained. Although not particularly limited, the polypeptide having RNA-dependent DNA polymerase activity may be a reverse transcriptase (RT) (EC 2.7.7.49). Although not particularly limited, reverse transcriptases from the lentivirus genus (including HIV, SIV, FIV, and EIA), alpharetrovirus genus (including avian myeloblastosis virus (AMV)), betaretrovirus genus, or gammaretrovirus genus (including Moloney murine leukemia virus (MMLV)) are preferred. Such reverse transcriptases may have the same amino acid sequence as the natural product, or may contain amino acid substitutions, deletions, and additions as long as activity is maintained. (i) napDNAbp and (ii) a polypeptide having RNA-dependent DNA polymerase activity may be fused via a linker. A nuclear localization signal, also known as a nuclear localization signal / sequence (NLS), is an amino acid sequence that serves as a marker for transporting a protein to the cell nucleus.Although not particularly limited, it is composed of one or more short sequences of positively charged amino acids (lysine, arginine). Such a sequence may be contained in the PrimeEditor enzyme and serve as a marker for transport to the cell nucleus, and may be located in the PrimeEditor enzyme sequence (NH. ２ The location of the nuclear localization signal does not matter (at the terminal, COOH-terminus, or internal to the enzyme). In one embodiment, a PrimeEditor enzyme containing such a nuclear localization signal may contain the amino acid sequence of PE2 (SEQ ID NO: 61), PE2* (SEQ ID NO: 62), or PEmax (SEQ ID NO: 30).

[0027] PrimeEditing gRNA containing a complementary strand sequence of a barcode sequence is an RNA containing a sequence encoding a spacer sequence, a gRNA core, a DNA synthesis template (including a complementary strand sequence of the barcode sequence), and a primer binding site in this order. The barcode sequence is a sequence containing any number of 5, 6, 7, or 8 bases. It may contain 0 to 4 bases of a predetermined base sequence and may be 5 to 12 bases in total length. PrimeEditing gRNA containing a complementary strand sequence of a barcode sequence can bind to the PrimeEditor enzyme and insert the barcode sequence into the target sequence described below through a Prime editing reaction. Although not particularly limited, the barcode sequence may be inserted in a manner that replaces a sequence of 1, 2, 3, 4, 5, 6, 7, or 8 consecutive bases within the target sequence. The combination of the pegRNA sequence and the following target sequence can be arbitrarily set based on the descriptions in Non-Patent Document 18 and Non-Patent Document 19.

[0028] In one embodiment of the present invention, (a) a fusion protein containing a PrimeEditor enzyme containing a nuclear localization signal may contain a first tag peptide or may be fused with the first tag peptide, and the extracellular vesicles may further contain (c) a protein that binds to a second tag peptide and the first tag peptide, wherein the binding affinity for the second tag peptide is greater than the binding affinity for the first tag peptide, and a fusion protein of a tag peptide-binding protein and a protein present in the extracellular vesicles. With this configuration, the PrimeEditor enzyme (a) encapsulating the PrimeEditing gRNA (b) is linked to a protein present in the extracellular vesicles via binding to the first tag peptide and the protein that binds to the tag peptide, thereby becoming localized in the extracellular vesicles. Alternatively, in one embodiment of the present invention, (a) the fusion protein containing the PrimeEditor enzyme containing a nuclear localization signal may contain a first antibody or may be fused to the first antibody, and the extracellular vesicles may further contain (c) a fusion protein of an antigenic peptide that binds to the first antibody and a protein present in the extracellular vesicles. With this configuration, the PrimeEditor enzyme (a) encapsulating the PrimeEditing gRNA (b) is linked to a protein present in the extracellular vesicles via the bond between the first antibody and the antigenic peptide, thereby becoming localized in the extracellular vesicles.

[0029] In the present invention, proteins present in extracellular vesicles are preferably extracellular vesicle markers, i.e., proteins whose detection proves the presence of (specific) extracellular vesicles (i.e., proteins that are abundantly present in extracellular vesicles or that are specifically present in extracellular vesicles). Their origin is not particularly limited, but they are preferably derived from humans, non-human mammals (including mice and rats), higher plants, or microorganisms (including enterobacteria).

[0030] According to Non-Patent Document 3, markers for mammalian extracellular vesicles are classified as follows. Membrane proteins or GPI-anchored proteins that can be used as marker proteins for extracellular vesicles include: 1) tissue-nonspecific tetraspanins (CD63, CD9, CD81, CD82), other multi-membrane spanning membrane proteins (CD47, heterotrimeric G proteins (GNA: Guanine nucleotide-binding proteins), etc.), MHC class I (HLA-A / B / C, H2-K / D / Q), integrins (ITGA / ITGB), transferrin receptor (TFR2); LAMP1 / 2; heparan sulfate proteoglycans (including syndecan (SDC)); extracellular matrix metalloproteinase inducer (EMMPRIN) (also known as BSG or CD147); ADAM10; CD73 (NT5E), a GPI-anchored 5'-nucleotidase; CD55 and CD59, GPI-anchored complement-binding proteins; Sonic Hedgehog protein (SHH); 2) Cell / tissue-specific: several tetraspanins: TSPAN8 (epithelial cell-specific), CD37 and CD53 (leukocyte-specific); PECAM1 (endothelial cell-specific); ERBB2 (breast cancer-specific); EPCAM (epithelial-specific); CD90 (THY1) (mesenchymal stem cell-specific); CD45 (PTPRC) (immune cell-specific), CD41 (ITGA2B) or CD42a (GP9) (platelet-specific); glycophorin A (GYPA) (erythrocyte-specific); CD14 (monocyte-specific), MHC class II (HLA-DR / DP / DQ, H2-A); Examples include CD3 (T cell specific); acetylcholinesterase / AChE-S (neuron specific), AChE-E (erythrocyte specific); amyloid βA4 / APP (neuron specific); and the like.

[0031] Cytoplasmic proteins that can be used as marker proteins for extracellular vesicles include ESCRT-I / II / III (TSG101, CHMP) and accessory proteins: ALIX (PDCD6IP), VPS4A / B; ARRDC1; flotillin-1 and 2 (FLOT1 / 2); caveolin (CAV); EHD; RHOA; annexin (ANXA); heat shock proteins HSC70 (HSPA8) and HSP84 (HSP90AB1); ARF6; syntenin (SDCBP); and microtubule-associated protein tau (Tau, MAPT; neuron-specific).

[0032] In addition, according to Non-Patent Document 16, LAMP2B, MFGE8, EWI immunoglobulin superfamily (IGSF8 and PTGFRN); and MARCKS protein family (MARCKS, MARKCSL1, and BASP1) can also be used as proteins present in extracellular vesicles in the present invention.

[0033] In the present invention, proteins present in extracellular vesicles may be naturally occurring proteins (including polymorphisms, orthologs, and paralogs), or may be artificial mutants in which some amino acids have been added, substituted, or deleted, or may be fragments thereof (for example, "exoTOPE" or 1 to 30 amino acid residues of BASP1), although artificial mutants or fragments that do not alter the localization of the protein are preferred.

[0034] In one embodiment of the present invention, the combination of the first tag peptide, the second tag peptide, and the tag-binding protein is preferably such that the first tag peptide and the second tag peptide can bind to and dissociate from the tag-binding protein in cells. More preferably, the binding affinity of the tag-binding protein for the second tag peptide is greater than the binding affinity for the first tag peptide. Although not particularly limited, such combinations may use any peptide (5 to 20 amino acid residues) as the first tag peptide; a single-chain antibody or a binding-active fragment thereof against the peptide as the tag-binding protein; and a peptide in which 1 to 10 amino acid substitutions, deletions, or additions have been introduced into the peptide to reduce its affinity with the single-chain antibody as the second tag peptide. In one embodiment, such combination is preferably the AlfaTag system described in Patent Document 6, in which the first tag peptide and the second tag peptide may comprise the amino acid sequence set forth in any of SEQ ID NOs: 64 to 177. Although not particularly limited, the first tag peptide may be an AlfaTag system. ＰＥ (SEQ ID NO: 21), and the second Tag peptide is AlfaTag ＳＴ (SEQ ID NO: 22). The tag peptide-binding protein is an anti-tag peptide single-chain antibody or a binding active fragment thereof, and preferably the tag-binding protein is an anti-Alfa Nanobody (SEQ ID NO: 27). Alternatively, in one embodiment, such a combination is preferably the SunTag system described in Non-Patent Document 17, in which a GNC peptide (SEQ ID NO: 23) or SunTag (SEQ ID NO: 24) is used as the second tag peptide, a GNC peptide or SunTag with one or two amino acid substitutions is used as the first tag peptide, and an anti-GCN antibody (SEQ ID NO: 25) can be used as the tag peptide-binding protein.

[0035] In one embodiment of the present invention, a combination of a first antibody and an antigenic peptide is preferably one in which the antigenic peptide can bind to and dissociate from the first antibody intracellularly. While not particularly limited, such a combination may use any peptide (5 to 20 amino acid residues) as the antigenic peptide, and a single-chain antibody against the peptide or a binding-active fragment thereof as the first antibody. In one embodiment, such a combination may include a combination of AlfaTag and an AlfaTag antibody in the AlfaTag system, or a combination of SunTag and a SunTag antibody.

[0036] In one embodiment of the present invention, extracellular vesicles contain a viral fusion protein or a fusion-active fragment thereof on their surface. Such enveloped viral fusion proteins can be classified into three types, Class I to Class III, based on their structural characteristics: Class I fusion proteins include influenza virus hemagglutinin (HA) and retrovirus Env; Class II fusion proteins include Semliki Forest virus (SFV) and dengue virus fusion proteins; and Class III fusion proteins include the G protein (VSV-G) of vesicular stomatitis virus (VSV), a rhabdovirus, and herpesvirus gB. Although not particularly limited, the viral fusion protein preferably contains VSG-G or a fusion-active fragment thereof. VSG-G may comprise the amino acid sequence of SEQ ID NO: 26, including embodiments having at least 75%, 80%, 90%, 95%, or 99% homology to the amino acid sequence of SEQ ID NO: 26 and maintaining its fusion activity. Alternatively, VSG-G may have 1 to 10 amino acid substitutions (e.g., K47Q, R354A, etc.) with respect to the amino acid sequence of SEQ ID NO: 26, so long as activity is maintained. Inclusion of a viral fusion protein or a fusion-active fragment thereof on its surface promotes fusion of extracellular vesicles with the cell membrane of their receptor cells, making it easier for extracellular vesicles (containing a fusion protein containing a PrimeEditor enzyme and a PrimeEditing gRNA) to be taken up into cells.

[0037] In one embodiment of the present invention, the barcode sequence is 5'NNNNNNTGNN3' (N is any base). In one embodiment of the present invention, (b) the PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 13 to 20 and 63:

[0038] The present invention includes a receiver cell that takes up the extracellular vesicles and records the barcode sequence. In one embodiment, the receiver cell is a cell that (i) contains in its nucleus a nucleic acid encoding a target sequence for a PrimeEditing gRNA; preferably, when the extracellular vesicles are taken up into the cell, (b) a fusion protein containing a PrimeEditor enzyme including a nuclear localization signal, which encapsulates a PrimeEditing gRNA including a complementary strand sequence of the barcode sequence, is translocated into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction. In one embodiment, such a receiver cell (i) contains in its nucleus a nucleic acid encoding a target sequence for a PrimeEditing gRNA; wherein, when the extracellular vesicle is taken up into the cell, the adhesion between the (c) Tag-binding protein and the (a) first Tag peptide is dissociated, resulting in (b) a fusion protein containing a PrimeEditor enzyme including a nuclear localization signal, which encapsulates a PrimeEditing gRNA including a complementary strand sequence of the barcode sequence, being translocated into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction; More preferably, the cell contains (ii) a fusion protein comprising the second Tag peptide in its cytoplasm, wherein the adhesion between the (c) Tag peptide-binding protein and the (a) first Tag peptide is dissociated by the antagonism of the fusion protein comprising the second Tag peptide (ii), resulting in (b) a fusion protein comprising the (a) first Tag peptide and a PrimeEditor enzyme comprising a nuclear localization signal, which harbors a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence, being translocated into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction.In one embodiment, such a receiver cell (i) comprises in its nucleus a nucleic acid encoding a target sequence for a PrimeEditing gRNA; wherein the adhesion between the antigen peptide of (c) and the first antibody of (a) is dissociated, resulting in (b) a fusion protein comprising a PrimeEditor enzyme including a nuclear localization signal, which harbors a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence, being translocated into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction; more preferably, the cell (ii) comprises in its cytoplasm a fusion protein comprising the first antibody, wherein the adhesion between the antigen peptide of (c) and the first antibody of (a) is dissociated due to the antagonism of the fusion protein comprising the first antibody of (ii), resulting in (b) a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence being translocated into the nucleus. (a) A fusion protein containing a gRNA, a first Tag peptide, and a PrimeEditor enzyme containing a nuclear localization signal is translocated into the nucleus, and a barcode sequence is inserted into the target sequence by a genome editing reaction.

[0039] The target sequence may be a sequence native to the receiver cell or may be an exogenously introduced sequence. Preferably, such a sequence is present in the cell (cytoplasm, nucleus, or genome) in the form of DNA and is not transient. In one embodiment, the target sequence includes, but is not limited to, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0040] In one embodiment, the receiver cell comprises a sequence encoding a fluorescent protein and / or a labeled protein, and the fluorescent protein or labeled protein is transcribed and translated only when the barcode sequence is inserted into the target sequence. For example, the sequence information is configured so that the ORF of the fluorescent protein and / or labeled protein is normal only when the barcode sequence is inserted into the target sequence. Alternatively, the sequence information is configured so that mRNA encoding the fluorescent protein and / or labeled protein is transcribed only when the barcode sequence is inserted into the target sequence.

[0041] In one embodiment, the receiver cell may be a HEK293 cell, a stem cell, an epithelial cell, an endothelial cell, a fibroblast, a cancer cell, an immune cell, a neuronal cell, or a plant cell.

[0042] The present invention includes donor cells that secrete the above-mentioned extracellular vesicles. In one embodiment, such donor cells comprise: (A) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal (a); and (B) a nucleic acid encoding a PrimeEditing gRNA containing a complementary strand sequence of the barcode sequence (b). The cells transcribe and translate the fusion protein (a) and transcribe the PrimeEditing gRNA (b). The transcribed and translated fusion protein (a) and PrimeEditing gRNA (b) form a complex within the cell, which is then incorporated into extracellular vesicles and secreted extracellularly.

[0043] In one embodiment, the donor cell comprises: (A) a nucleic acid encoding a fusion protein comprising the first tag peptide of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) a nucleic acid encoding a fusion protein of a tag peptide-binding protein present in extracellular vesicles and a protein that binds to the second tag peptide of (c), the tag peptide-binding protein having a greater binding affinity to the second tag peptide than to the first tag peptide. The cell transcribes and translates the fusion protein of (a) and the fusion protein of (c), and transcribes the PrimeEditing gRNA of (b). The transcribed and translated fusion protein (a) and PrimeEditing gRNA (b) form a complex within the cell, and by linking with the fusion protein (c), the complex is specifically incorporated into extracellular vesicles and secreted outside the cell.

[0044] In one embodiment, the donor cell comprises: (A) a nucleic acid encoding a fusion protein comprising the first antibody (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence (b); and (C) a nucleic acid encoding a fusion protein of an antigen peptide that binds to the first antibody (c) and a protein present in extracellular vesicles. The cell transcribes and translates the fusion protein (a) and the fusion protein (c), and transcribes the PrimeEditing gRNA (b). The transcribed and translated fusion protein (a) and PrimeEditing gRNA (b) form a complex within the cell, which is then linked to the fusion protein (c) and specifically incorporated into extracellular vesicles and secreted outside the cell.

[0045] In one embodiment, the donor cell further comprises (D) a nucleic acid encoding a viral fusion protein (d). The donor cell transcribes and translates the viral fusion protein (d), and the transcribed and translated viral fusion protein (d) is displayed on the outer surface of the secreted extracellular vesicles, thereby promoting adhesion between the extracellular vesicles and receiver cells.

[0046] In one embodiment, in such donor cells, a fusion protein of (C) and a protein present in the extracellular vesicles of (c) contains a peptide corresponding to the barcode sequence and is presented outside the vesicles. The barcode sequence and the peptide do not have to have a one-to-one relationship, and may have a one-to-multiple relationship, i.e., multiple types of peptides are linked to one type of barcode sequence.

[0047] In one embodiment, such donor cells contain nucleic acids that affect the properties of the extracellular vesicles.

[0048] In the present disclosure, nucleic acids that affect the properties of extracellular vesicles include nucleic acids that, when analyzed, result in the following changes: (1) a change in the amount of endogenous proteins present within or on the surface of the secreted extracellular vesicles compared to when the cells secreting the extracellular vesicles contain the nucleic acid; (2) a change in the amount of extracellular vesicles secreted compared to when the cells secreting the extracellular vesicles do not contain the nucleic acid; (3) an effect on the lipid membrane that constitutes the extracellular vesicle membrane compared to when the cells secreting the extracellular vesicles do not contain the nucleic acid; (4) the presence of an exogenous protein encoded by the nucleic acid within or on the surface of the extracellular vesicles. The nucleic acid may be naturally occurring DNA or RNA, or a mixture thereof. Non-naturally occurring nucleic acids (e.g., nucleic acids in which some or all of the nucleotides are bonded via phosphorylated esters (P) rather than via phosphorylation, or peptide nucleic acids (PNAs)) may also be used.

[0049] (1) Examples of the nucleic acid encoding an endogenous protein itself, a nucleic acid encoding a transcription factor that controls the transcription of an endogenous protein, a nucleic acid encoding a factor involved in the post-translational modification of an endogenous protein, a nucleic acid encoding a factor involved in the chaperoning of an endogenous protein (including folding and intracellular transport), and other nucleic acids encoding proteins; antisense RNA (siRNA), miRNA (microRNA), shRNA (small hairpin RNA), and snRNA (small nuclear RNA) that positively or negatively regulate the expression of an endogenous protein, or a nucleic acid for modifying a gene encoding an endogenous protein using genome editing technology (e.g., ZFN (Zinc-Finger Nuclease), TALEN (Transcription Activator-Like Effector Nuclease), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 (Crispr Associated protein 9), etc.) (e.g., gRNA used in the CRISPR / Cas9 system), and the like. (2) Examples of such nucleic acids include nucleic acids encoding factors that affect the amount of extracellular vesicles secreted, and nucleic acids encoding factors that control the transcription, translation, and expression of factors that affect the amount of extracellular vesicles secreted themselves; antisense RNA, miRNA, shRNA, and snRNA that positively or negatively regulate the expression of factors that control the transcription, translation, and expression of factors that affect the amount of extracellular vesicles secreted or factors that affect the amount of extracellular vesicles secreted themselves, or nucleic acids for modifying genes that encode factors that control the transcription, translation, and expression of factors that affect the amount of extracellular vesicles secreted or factors that affect the amount of extracellular vesicles secreted themselves in genome editing technology (e.g., ZFN, TALEN, CRISPR / Cas9) (e.g., gRNA used in the CRISPR / Cas9 system).(3) Examples include nucleic acids encoding enzymes for synthesizing the lipid membrane that constitutes the extracellular vesicle membrane, nucleic acids encoding factors for controlling the transcription, translation, and expression of enzymes for synthesizing the lipid membrane that constitutes the extracellular vesicle membrane; antisense RNA, miRNA, shRNA, and snRNA that negatively or positively control the expression of factors for controlling the transcription, translation, and expression of enzymes for synthesizing the lipid membrane that constitutes the extracellular vesicle membrane, or enzymes for synthesizing the lipid membrane that constitutes the extracellular vesicle membrane in genome editing technology (e.g., ZFN, TALEN, CRISPR / Cas9, etc.), enzymes for synthesizing the lipid membrane that constitutes the extracellular vesicle membrane, and enzymes for synthesizing the lipid membrane that constitutes the extracellular vesicle membrane, nucleic acids for modifying genes encoding factors for controlling the transcription, translation, and expression of enzymes for synthesizing the lipid membrane that constitutes the extracellular vesicle membrane (e.g., gRNA used in the CRISPR / Cas9 system), etc. Examples of (4) include nucleic acids encoding exogenous proteins.

[0050] In the present disclosure, nucleic acids that affect the properties of extracellular vesicles may include mRNA and ncRNA.

[0051] In the present disclosure, mRNA refers to RNA including RNA (cRNA; coding RNA) having base sequence information and a structure that can be translated into a protein (or peptide), and includes not only naturally occurring mRNA but also RNA that does not contain an m7G cap at the 5' end or a polyadenylation (polyA) at the 3' end. It also includes premature mRNA that, if properly spliced ​​in cells, will have the base sequence information and structure that can be translated into a protein.

[0052] Non-coding RNA (ncRNA) refers to RNA (functional nucleic acid) that does not have the base sequence information and structure that can be translated into protein, but has some function in the body. It includes, but is not limited to, small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA), which form complexes with proteins in the nucleus, as well as miRNA (including pre-miRNA) and siRNA (including pre-siRNA (e.g., shRNA (small hairpin RNA))) that bind to other RNA. Furthermore, ncRNA may also include guide RNA (gRNA; including single-stranded guide RNA) (RNA that guides an RNA:protein complex to a target nucleic acid molecule through complementary binding). In the CRISPR / Cas9 system in bacteria and archaea, gRNA is a combination of two types of RNA: crRNA (CRISPR RNA), which recognizes a target DNA sequence of approximately 20 bases, and tracrRNA (trans-activating crRNA), which acts as a scaffold for binding to Cas9. However, gRNA also includes sgRNA (single guide RNA), which combines these two RNAs for the purpose of genome editing.

[0053] When using a guide RNA as a nucleic acid that affects the properties of extracellular vesicles, it is preferable to express a nucleic acid-programmed DNA-binding protein different from the i) nucleic acid-programmed DNA-binding protein (napDNAbp) contained in the PrimeEditor enzyme. For example, when the i) nucleic acid-programmed DNA-binding protein (napDNAbp) contained in the PrimeEditor enzyme is Cas9, it is preferable to express Cas12a (Cpf1) endonuclease. When Cpf1 is used, only the 41-44 base crRNA (recognition region is 21-24 bases) functions as gRNA. The nucleic acid-programmed DNA-binding protein and the guide RNA that affect the properties of extracellular vesicles modify the genes of the donor cell, thereby affecting the properties of the extracellular vesicles secreted by the cell.

[0054] The donor cells of the present invention are not particularly limited as long as they are derived from eukaryotes, but are preferably derived from humans, non-human mammals (including mice and rats), or higher plants. Mammalian cells include stem cells (including induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and somatic stem cells (including mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, neural stem cells, vascular endothelial stem cells, hepatic stem cells, and epithelial stem cells)), cells induced to differentiate from stem cells, epithelial cells, endothelial cells, fibroblasts, cancer cells, immune cells (dendritic cells and blood cells), and nerve cells, as well as established cell lines of these cells. Cultured cells (e.g., HEK293T cells) are also acceptable.

[0055] The present invention includes nucleic acids for producing the donor cells. In one embodiment, such nucleic acids include: (A1) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme comprising a nuclear localization signal of (a); (A2) a nucleic acid encoding a fusion protein comprising the first Tag peptide of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (A3) a nucleic acid encoding a fusion protein comprising the first antibody of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); (C1) a nucleic acid encoding a fusion protein of a Tag peptide-binding protein present in extracellular vesicles and a protein that binds to the second Tag peptide of (c), the protein having a higher binding affinity to the second Tag peptide than to the first Tag peptide; and / or (C2) A nucleic acid encoding a fusion protein of an antigen peptide that binds to the first antibody of (c) and a protein present in extracellular vesicles. A1 to 3, C1, and C2 are used to transcribe and translate the fusion protein, and B is used to transcribe Prime Editing gRNA. The nucleic acid may be linked to an appropriate promoter sequence for such transcription and translation. The nucleic acid may be included in an expression vector for transcription and translation in donor cells.

[0056] In one embodiment, the nucleic acid comprises (D) a nucleic acid encoding the viral fusion protein (d). D is used to transcribe and translate the viral fusion protein. The nucleic acid may be linked to an appropriate promoter sequence for such transcription and translation, or the nucleic acid may be included in an expression vector for transcription and translation in the donor cell.

[0057] In one embodiment, such nucleic acids include (E) nucleic acids that affect the properties of extracellular vesicles. The nucleic acid may be linked to an appropriate promoter sequence for such transcription and translation, or may be included in an expression vector for transcription and translation in donor cells. The nucleic acids that affect the properties of extracellular vesicles include: (1) nucleic acids that change the amount of endogenous proteins present within or on the surface of extracellular vesicles; (2) nucleic acids that promote or inhibit the secretion of extracellular vesicles; (3) nucleic acids that affect the lipid membrane that constitutes the membrane of extracellular vesicles; and (4) nucleic acids that cause exogenous proteins to be present within or on the surface of extracellular vesicles, including mRNA or ncRNA (including miRNA, siRNA, shRNA, snRNA, snoRNA, gRNA, and sgRNA).

[0058] In one embodiment, such nucleic acids include expression vectors for expressing nucleic acid programmable DNA binding proteins (napDNAbp).

[0059] The present invention includes nucleic acids for producing the receiver cells. In one embodiment, such nucleic acids include (i) a nucleic acid encoding a target sequence for a PrimeEditing gRNA; and / or (ii) a nucleic acid encoding a fusion protein including a second Tag peptide. (i) The nucleic acid encoding the target sequence for a PrimeEditing gRNA may further include a sequence encoding a fluorescent protein and / or a labeling protein, and may be configured so that the fluorescent protein or labeling protein is transcribed and translated only when the barcode sequence is inserted into the target sequence. The nucleic acids (i) and (ii) may be contained in an expression vector.

[0060] The present invention includes non-human organisms that contain or consist of the receiver cells. The non-human organisms may be plants, animals (including mice and rats), or microorganisms. Such organisms may be produced by transplanting the receiver cells into an organism, or may be produced by known methods for producing transgenic animals using nucleic acids for producing the receiver cells.

[0061] The present invention includes a method for producing a library of the above-mentioned extracellular vesicles. In one embodiment, the method includes: 1) introducing into an extracellular vesicle-secreting cell: (i) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme comprising a nuclear localization signal, or an expression vector for expressing the fusion protein; and (ii) a combination of: a1) a nucleic acid comprising a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence of at least one barcode sequence; and a2) a nucleic acid comprising at least one sequence that affects the properties of extracellular vesicles corresponding to the barcode sequence; or a combination of: a) a nucleic acid comprising a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence of at least one barcode sequence; and at least one sequence that affects the properties of extracellular vesicles corresponding to the barcode sequence; 2) culturing the extracellular vesicle-secreting cells after the introduction in a culture medium; and 3) isolating the fusion protein comprising the PrimeEditor enzyme and the PrimeEditing gRNA from the culture supernatant of the extracellular vesicle-secreting cells. and recovering the extracellular vesicles containing the gRNA. In such a method, the extracellular vesicle-secreting cells may express Cas12a (Cpf1) endonuclease, and the sequence that affects the properties of the extracellular vesicles may be a sequence of a gRNA or sgRNA for an endogenous gene.

[0062] The present invention includes a method for producing a library of extracellular vesicles that display peptides on their outer surface. In one embodiment, the method includes: 1) introducing into an extracellular vesicle-secreting cell: (i) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal, or an expression vector for expressing the fusion protein; and (ii) a combination of: a) a nucleic acid comprising a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence to at least one barcode sequence; and b) a nucleic acid comprising a sequence encoding a fusion protein comprising a peptide corresponding to the barcode sequence and a protein present in the extracellular vesicles to be presented outside the vesicles; or a combination of: a1) a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence to at least one barcode sequence; and a2) a sequence encoding a fusion protein comprising a peptide corresponding to the barcode sequence and a protein present in the extracellular vesicles to be presented outside the vesicles; 2) culturing the extracellular vesicle-secreting cell after the introduction in a culture medium; and 3) recovering extracellular vesicles that contain the fusion protein containing the PrimeEditor enzyme and the PrimeEditing gRNA and present the peptide from the culture supernatant of the extracellular vesicle-secreting cells.

[0063] the method further comprises, before step 2), a step of introducing a nucleic acid comprising a sequence encoding a fusion protein of a tag peptide-binding protein that binds to a second tag peptide and the first tag peptide, the tag peptide-binding protein having a higher binding affinity to the second tag peptide than the first tag peptide, and a protein present in extracellular vesicles, wherein the fusion protein comprising the Prime Editor enzyme comprising a nuclear localization signal of (i) may comprise the first tag peptide; or, before step 2), a step of introducing a nucleic acid comprising a sequence encoding a fusion protein of a tag peptide-binding protein that binds to a second tag peptide and the first tag peptide, the tag peptide-binding protein having a higher binding affinity to the second tag peptide than the first tag peptide, and a protein present in extracellular vesicles, wherein the fusion protein comprising the Prime Editor enzyme comprising a nuclear localization signal of (i) may comprise the first tag peptide; Here, the first tag peptide may comprise AlfaTagPE (SEQ ID NO: 21), the second tag peptide may comprise AlfaTagST (SEQ ID NO: 22), and the tag-binding protein may comprise an anti-Alfa nanobody (SEQ ID NO: 27).

[0064] The above method may further comprise, before step 2), a step of introducing a nucleic acid comprising a sequence encoding a fusion protein of an antigen peptide that binds to the first antibody and a protein present in extracellular vesicles, wherein the fusion protein comprising the PrimeEditor enzyme containing a nuclear localization signal of (i) may comprise the first antibody; wherein the antigen peptide may be SUNtag peptide (GNC peptide x10) (SEQ ID NO: 24), and the first antibody may be an anti-GCN antibody (SEQ ID NO: 25).

[0065] The above method may further comprise, before step 2), a step (d) of introducing a nucleic acid comprising a sequence encoding a viral fusion protein.

[0066] In the above method, the protein present in the extracellular vesicles may be CD9 (SEQ ID NO: 28) or BASP1 (1-30) (SEQ ID NO: 29); the barcode sequence may comprise any nucleic acid sequence of 5 to 8 bases; the barcode sequence may be 5'NNNNNNTGNN3' (N is any base); and / or the PrimeEditing gRNA has a sequence selected from the group consisting of SEQ ID NOs: 13 to 20 and 63: may include:

[0067] In one embodiment of the present invention, in a method for producing an extracellular vesicle library, the extracellular vesicle library contains at least two or more types of extracellular vesicles, and the extracellular vesicles are identified by determining the sequence of the barcode RNA encapsulated therein. Preferably, the library contains 5,000 or more types, 6,000 or more types, 7,000 or more types, 8,000 or more types, 9,000 or more types, or 10,000 or more types of extracellular vesicles. When recovering extracellular vesicles, a specific extracellular vesicle marker may be used as an indicator to recover only specific types of extracellular vesicles. For example, an antibody that recognizes a membrane protein, such as a tetraspanin, present in the membrane of the extracellular vesicles can be used to recover only extracellular vesicles that have the tetraspanin on their membrane surface without disruption.

[0068] The library of extracellular vesicles can be added to the receiver cells, and the barcode sequences taken up by the receiver cells can be used to determine which extracellular vesicles have been taken up by the receiver cells. Alternatively, the library of extracellular vesicles can be administered to a non-human organism containing / consisting of the receiver cells, and the barcode sequences taken up by the receiver cells can be used to determine which extracellular vesicles have been taken up by which tissue-containing receiver cells. Primary screening to determine whether or not the extracellular vesicles have been taken up can be performed by PCR or the like, or by treating extracted DNA from the receiver cells with a target sequence-specific restriction enzyme (the restriction enzyme recognition site is lost upon insertion of the barcode sequence). Alternatively, if the fluorescent protein or labeled protein is transcribed and translated only when the barcode sequence is inserted, detection of the fluorescent protein or labeled protein can be performed. Secondary screening to determine which extracellular vesicles have been taken up can be performed by sequencing the sequence of the target sequence region.

[0069] The above-mentioned extracellular vesicle library can be used for the following purposes: (1) identification of factors involved in changes in the secretion amount of extracellular vesicles (including factors involved in changes in the secretion amount of vesicles containing specific proteins); (2) identification of factors involved in the localization of specific proteins in / on the membrane of extracellular vesicles; (3) identification of factors affecting the half-life and dynamics of extracellular vesicles in body fluids; (4) identification of factors affecting the targeting of extracellular vesicles to each tissue or each body fluid; (5) identification of factors affecting the targeting of extracellular vesicles to specific cells (including primary cultured cells); etc.

[0070] Example 1. Selection of target sequence A. Construct for target sequence (mCherry-T2A-target-GFP-E2A-luciferase) Figure 1A shows a schematic diagram of the construct introduced into receiver cells. The following target sequences #1 to #6 were used. The target sequences were designed to contain the following BcgI or BsaXI restriction enzyme recognition sequences (restriction enzyme recognition sequences are shown in lowercase). The sequence information for a construct in which #5 has been inserted as the target sequence in SEQ ID NO: 33 is shown as an example. The designed construct was inserted into the SfiI site of pSBbi-Hyg (addgene #60524) to create an expression vector. PrimeEditing was used to insert and / or replace the barcode sequence targeting the underlined base (inserting five bases before the underlined base). Only when the barcode sequence was replaced did the fluorescent protein (GFP) and marker protein (luciferase) downstream of the target sequence be translated and expressed normally.

[0071] B. PEGRNA Expression Constructs Constructs capable of transcribing the following PEGRNAs #1 to #6 were prepared in a manner corresponding to the target sequences #1 to #6 above. The vector plasmid for transcribing PEGRNA was prepared by introducing the sequence encoding PEGRNA into pU6-PEGRNA-GG-acceptor (addgene #132777). The double-underlined base sequence corresponds to the complementary strand sequence of the barcode sequence to be inserted into the target sequence (ie, in this example, 5'-AGGAA-3' is inserted into the target sequence).

[0072] C. PEmax Expression Construct A sequence encoding PEmax (SEQ ID NO: 30) was inserted into the SfiI site of pSBbi-Hyg (addgene #60524) to prepare an expression vector.

[0073] A) Constructs for each target sequence, B) the corresponding PEGRNA expression plasmid, and C) the Prime Editor expression plasmid were transiently transfected into HEK293T cells. Two days after transfection, the cells were detached and mCherry and GFP expression was analyzed using a flow cytometer. D-luciferin was also added to the cells, and bioluminescence emitted from the cells was monitored using a plate reader. The results are shown in Figure 1B. All of the target sequence and PEGRNA combinations used (#1 to #6) demonstrated a PEGRNA-dependent PrimeEdit reaction, resulting in the detection of GFP and luciferase activity.

[0074] Example 2. Optimization of pegRNA Sequence The sequence of pegRNA #5 was adjusted to optimize the efficiency of the PrimeEdit reaction for the target sequence #5. The spacer, scaffold variant, RT template length, and the presence or absence of a 3' motif were examined (Figure 2A). The sequences of the pegRNAs actually used are shown below. The double-underlined base sequence corresponds to the complementary strand sequence of the barcode sequence to be inserted into the target sequence.

[0075] Using HEK293T cells as parent cells, a stable cell line containing target sequence #5 was generated using Sleeping Beauty transposase. The optimized pegRNA, Prime Editor (PEmax), was then transiently transfected into the cell as described in Example 1. Two days after transfection, D-luciferin was added, and bioluminescence emitted from the cells was monitored using a plate reader. The relative editing efficiency calculated from the bioluminescence values ​​is shown in Figure 2B. The PrimeEdit reaction occurred regardless of the pegRNA used.

[0076] Example 3. Uptake of PEG RNA into Extracellular Secretory Vesicles via the AlFaTag System and SunTag System The AlFaTag system and SunTag system were introduced to allow PE (+PEG RNA) to dissociate within receiver cells, rather than directly linking PE (+PEG RNA) to proteins present in extracellular vesicles (Figure 3A). A. Expression Constructs 1. CD9-antiALFANb A vector plasmid expressing the CD9-anti-ALFA Nanobody fusion protein (SEQ ID NO: 40) was prepared by introducing a sequence encoding the CD9-anti-ALFA Nanobody fusion protein into the CMV promoter of pcDNA3.1(+). 2. CD9-SunTag A vector plasmid expressing the CD9-SunTag fusion protein (SEQ ID NO: 41) was prepared by introducing a sequence encoding the protein into the CMV promoter of pcDNA3.1(+). 3. 3. BASP1-antiALFANb A vector plasmid expressing the BASP1-anti-ALFA nanobody fusion protein (SEQ ID NO: 42) was prepared by introducing a sequence encoding the protein into the CMV promoter of pcDNA3.1(+). 4. BASP1-SunTag A vector plasmid expressing the BASP1-SunTag fusion protein (SEQ ID NO: 43) was prepared by introducing a sequence encoding the protein into the CMV promoter of pcDNA3.1(+). 5. AlfaTag-PE A vector plasmid expressing the AlfaTag-PrimeEditor fusion protein (SEQ ID NO: 44) was prepared by inserting a sequence encoding the protein into the SfiI site of pSBbi-Hyg (addgene #60524). 6. A vector plasmid expressing the anti-GCNscFv-PE anti-SunTag antibody-PrimeEditor fusion protein (SEQ ID NO: 45) was prepared by inserting a sequence encoding the protein into the SfiI site of pSBbi-Hyg (addgene #60524).7. CD9-PE A vector plasmid expressing a fusion protein (SEQ ID NO: 46) in which CD9 and PrimeEditor are fused via a TEV linker was prepared by inserting a sequence encoding the protein into the SfiI site of pSBbi-Hyg (addgene #60524). 8. PEG RNA A vector plasmid for transcribing PEG RNA (SEQ ID NO: 47) was prepared by introducing a sequence encoding PEG RNA into pU6-PEGRNA-GG-acceptor (addgene #132777).

[0077] B. EV Production HEK293T cells cultured in 24 wells / plate were transfected with the above-mentioned combination of fusion protein expression vectors and pegRNA transcription vector in the following combinations and ratios. Control cells: Transiently expressed only the pegRNA expression plasmid (pSY62 AGGAA insertion). CD9-PE (TEV cleavable): Transiently expressed the CD9-PE expression plasmid and pegRNA expression plasmid. SUNtag-system: Transiently expressed anchor (CD9-SUNtag or BASP1 (1-30)-SUNtag), a fusion of Prime Editor and anti-GCN antibody, and pegRNA expression plasmid. ALFAtag system: Transiently expressed anchor (CD9-antiALFA nanobody or BASP1 (1-30)-SUNtag), a fusion of Prime Editor and ALFA tag (ALFA(ST)), and pegRNA expression plasmid. One day after transfection, the medium was replaced, and the supernatant was collected 48 hours after the replacement. After removing contaminants from the supernatant using a filter, RNA was extracted from the supernatant, and the amount of pegRNA was quantified by qPCR.

[0078] The results are shown in Figure 3B, which demonstrates that pegRNA could be incorporated into EVs in both the AlFaTag system and the SunTag system.

[0079] Example 4. Effect of antagonistic Tag peptide and viral fusion protein on editing efficiency To improve editing efficiency, a viral fusion protein was expressed in extracellular vesicles and an antagonistic Tag peptide was expressed in receiver cells. The viral fusion protein (VSV-G) promoted the fusion of extracellular vesicles with receiver cells, and the antagonistic Tag peptide (AlFaTag) ＳＴ ) facilitates the dissociation of intracellularly incorporated PE (+pegRNA) from a protein (CD9) present in extracellular vesicles, thereby promoting nuclear translocation (Figure 4A).

[0080] A. Constructs for extracellular vesicles 1. CD9-antiALFANb A vector plasmid expressing the CD9-anti-ALFA nanobody fusion protein (SEQ ID NO: 40) was prepared by introducing a sequence encoding the CD9-anti-ALFA nanobody fusion protein into the EF-1α promoter of the pSBbi-GH vector (addgene, plasmid #60514; hygromycin resistance gene + EGFP co-expression type). 2. AlfaTag ＰＥ -PE AlfaTag ＰＥ A vector plasmid expressing the PrimeEditor fusion protein (SEQ ID NO: 44) was prepared by inserting a sequence encoding the protein into the SfiI site of pSBbi-Hyg (addgene #60524). 3. PEG RNA: Each vector plasmid transcribing PEG RNA (SEQ ID NO: 63) (pRK455 (barcode: AGGAAT); pSY115 (barcode: CATTCA); pSY117 (barcode: GCCTAA), pSY120 (barcode: TGTCGA)) was prepared by introducing a sequence encoding PEG RNA into pU6-tevopreq1-GG-acceptorp (addgene #174038). 4. VSV-G: pMD2.G (Addgene #12259) was used to express VSV-G (SEQ ID NO: 26).

[0081] B. Establishment of receiver cell line 1. mCherry-T2A-target-GFP-E2A-luciferase (SEQ ID NO: 33) A construct was created that stably expresses mCherry (a fluorescent protein) and expresses GFP and luciferase only when a barcode sequence is inserted into target sequence #5. HEK293T cells were used as parent cells, and a stable cell line containing the above construct was created using sleeping beauty transposase. 2. AlFa ST mCherry-T2A-target-GFP-E2A-luciferase (SEQ ID NO: 49) Alfa ＳＴ We created a construct that stably expresses fusion mCherry and expresses GFP and luciferase only when the barcode sequence is inserted into the target sequence. We used HEK293T cells as parent cells and generated a stable cell line containing the construct using sleeping beauty transposase.

[0082] C1. EV production CD9-antiALFANb, AlfaTag ＰＥ EV-producing cells were transfected with PE and pegRNA (pRK455 (AGGAAT)), and VSV-G (VSV-G only; if no VSV-G was present, the same amount of filler plasmid (pcDNA3.1) was used). The medium was changed one day after transfection. Two days after the medium change, the EV-containing supernatant was collected and contaminants were removed using a filter. D1. PrimeEdit reaction The filter-treated supernatant prepared in C1 was applied to HEK293T cells stably expressing the reporter prepared in B. After 72 hours of culture, the cells were collected, detached, and mCherry and GFP fluorescence was measured using a flowcytometer (Figure 4B).

[0083] C2. EV production CD9-antiALFANb, AlfaTag ＰＥEV-producing cells were transfected with PE and pegRNA, and VSV-G (VSV-G only, or without VSV-G, with the same amount of filler plasmid (pcDNA3.1)). The medium was changed one day after transfection. Two days after the medium change, the EV-containing supernatant was collected and contaminants were removed using a filter. In the presence of VSV-G, A) pRK455 (AGGAAT) or B) pSY107 (AGGACA) was used; in the absence of VSV-G, C) pSY115 (CATTCA), D) pSY117 (GCCTAA) or E) pSY120 (TGTCGA) were used as expression vectors for pegRNA. D2. The filter-treated supernatant prepared in PrimeEdit reaction C2 was applied to HEK293T cells stably expressing the reporter prepared in B. After 72 hours of culture, the cells were collected, detached, and genomic DNA was extracted. Using this as a template, the target sequence region was amplified by PCR, treated with BsaXI, and the unedited target sequence was removed. The amount of barcode was then quantified by qPCR (Figure 4C).

[0084] 4B and 4C show the viral fusion protein (VSV-G) and the first Tag peptide (Alfa ＰＥ ) and a second Tag peptide (Alfa ＳＴ ) promotes the PrimeEdit reaction via EV.

[0085] Example 5. Insertion of Longer Barcode Sequences We investigated the length of the barcode sequence to be inserted into the target sequence. Typically, a single base in the target sequence (e.g., the underlined c in the #5 target sequence: TgAATTAGCTACCTGTGAGAa-c-TGTGGctccGTTGGCGTCCGAAGCT) is inserted into the target sequence, but we investigated whether a barcode sequence could be inserted by including several bases downstream of that base (Figure 5A). A vector plasmid for transcribing the following PEG RNA (SEQ ID NOS: 50-60) capable of inserting the mixed base sequence was constructed using the addgene 174038 pU6-tevopreq1-GG acceptor as a backbone, encoding the PEG RNA under the U6 promoter. The #19 (SEQ ID NOS: 37) used in Example 2 was used as a control.

[0086] Using HEK293T cells as parent cells, a stable cell line containing target sequence #5 was generated using Sleeping Beauty transposase. The above-mentioned pegRNA expression vector and Prime Editor (PEmax) expression vector were transiently transfected into the cells as described in Example 2. Two days after transfection, D-luciferin was added, and bioluminescence emitted from the cells was monitored using a plate reader. The results are shown in Figure 5B. The PrimeEdit reaction occurred regardless of the pegRNA used.

[0087] Other sequence information used in the examples is shown below.

[0088] Use of the extracellular vesicle fate recording system according to the present invention can be useful in the development of an efficient drug delivery system using exosomes, in exosome biology research (for example, elucidating EV secretion in various cells), and in drug discovery research targeting the exosome secretion pathway (for example, identifying factors that change the amount of EV secretion in a cell-specific manner).

[0089] The present invention can also be used to analyze exosome-mediated networks across biological kingdoms. For example, in the mammalian digestive tract, in addition to plants (food), intestinal bacteria, pathogenic microorganisms, and dietary yeasts constantly interact with each other, and this method can be useful for studying the exosomes produced by these heterogeneous biological communities.

Claims

1. (a) a fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal; and (b) PrimeEditing gRNA containing the complementary strand sequence of the barcode sequence containing extracellular vesicles.

2. (a) a fusion protein comprising a first Tag peptide and a PrimeEditor enzyme comprising a nuclear localization signal; (b) a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence; and (c) A fusion protein of a tag peptide-binding protein and a protein present in extracellular vesicles, the tag peptide-binding protein binding to a second tag peptide and the first tag peptide, wherein the binding affinity to the second tag peptide is greater than the binding affinity to the first tag peptide. The extracellular vesicle of claim 1, comprising:

3. The extracellular vesicle of claim 2, wherein the first tag peptide comprises AlfaTagPE (SEQ ID NO: 21), the second tag peptide comprises AlfaTagST (SEQ ID NO: 22), and the tag-binding protein comprises an anti-Alfa nanobody (SEQ ID NO: 27).

4. (a) a fusion protein comprising a first antibody and a PrimeEditor enzyme containing a nuclear localization signal; (b) a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence; and (c) a fusion protein of an antigen peptide that binds to the first antibody and a protein present in extracellular vesicles. The extracellular vesicle of claim 1, comprising:

5. The extracellular vesicle of claim 4, wherein the antigen peptide is SUNtag peptide (GNC peptide x10) (SEQ ID NO: 24) and the first antibody is an anti-GCN antibody (SEQ ID NO: 25).

6. The extracellular vesicle of claim 1, further comprising (d) a viral fusion protein on its surface.

7. (d) The extracellular vesicle of claim 6, wherein the viral fusion protein comprises VSV-G.

8. The extracellular vesicle of claim 1, wherein the protein present in the extracellular vesicle comprises CD9 (SEQ ID NO: 28) or BASP1 (1-30) (SEQ ID NO: 29).

9. The extracellular vesicle according to claim 2, wherein the protein present in the extracellular vesicle (c) further contains a peptide corresponding to a barcode sequence and is displayed outside the vesicle.

10. The extracellular vesicle of claim 1, wherein the barcode sequence comprises any nucleic acid sequence of 5 to 8 bases.

11. The barcode sequence is The extracellular vesicle of claim 10, wherein the amino acid sequence is 5'NNNNNNNTGNN3' (wherein N is any base).

12. The extracellular vesicle of claim 10, wherein the Prime Editing gRNA comprising a complementary strand sequence of the barcode sequence (b) comprises a sequence selected from the group consisting of SEQ ID NOs: 13 to 20 and 63: 【Table 12】

13. When the extracellular vesicle according to claim 1 is endocytosed into a cell, A fusion protein containing a PrimeEditor enzyme including a nuclear localization signal (a) encapsulating a PrimeEditing gRNA including a complementary strand sequence of the (b) barcode sequence is translocated into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction. (i) A cell containing a nucleic acid encoding a target sequence for the Prime Editing gRNA in its nucleus.

14. When the extracellular vesicle according to claim 2 is taken up into a cell, The adhesion between the (c) tag peptide-binding protein and the (a) fusion protein comprising the first tag peptide and a PrimeEditor enzyme comprising a nuclear localization signal is dissociated by the antagonism with the (ii) fusion protein comprising the second tag peptide, and as a result, the (b) fusion protein comprising the (a) first tag peptide and a PrimeEditor enzyme comprising a nuclear localization signal, which encapsulates a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence, is translocated into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction. (i) containing a nucleic acid encoding a target sequence for the Prime Editing gRNA in the nucleus; and (ii) a fusion protein containing the second Tag peptide is contained in the cytoplasm. cell.

15. The first Tag peptide is AlfaTag PE (SEQ ID NO: 21), and the second Tag peptide is AlfaTag ST (SEQ ID NO: 22) and the Tag-binding protein comprises an anti-Alfa Nanobody (SEQ ID NO: 27).

16. When the extracellular vesicle-secreting cell according to claim 4 is taken up into a cell, The adhesion between the antigen peptide of (c) and the first antibody of (a) is dissociated, and as a result, a fusion protein containing a PrimeEditor enzyme including a nuclear localization signal (a) encapsulating a PrimeEditing gRNA including a complementary strand sequence of the barcode sequence (b) is translocated into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction. (i) A cell comprising a nucleic acid encoding a target sequence for the Prime Editing gRNA in its nucleus.

17. The target sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 6: 【Table 13】 The cell of claim 13.

18. The cell of claim 13, comprising a sequence encoding a fluorescent protein and / or a label protein, wherein the fluorescent protein or label protein is transcribed and translated only when the barcode sequence is inserted within a target sequence.

19. The cell according to any one of claims 13 to 18, which is selected from the group consisting of HEK293 cells, stem cells, epithelial cells, endothelial cells, fibroblasts, cancer cells, immune cells, neural cells and plant cells.

20. A cell that secretes the extracellular vesicles of claim 1, (A) a nucleic acid encoding a fusion protein comprising the PrimeEditor enzyme containing the nuclear localization signal of (a); and (B) A nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b). including, cells.

21. The cell secreting the extracellular vesicles of claim 2, (A) a nucleic acid encoding a fusion protein comprising the first Tag peptide of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) A nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) A nucleic acid encoding a fusion protein of a tag peptide-binding protein that binds to the second tag peptide of (c) and the first tag peptide, wherein the binding affinity for the second tag peptide is greater than the binding affinity for the first tag peptide, and a protein present in extracellular vesicles. Extracellular vesicle-secreting cells, including

22. The cell secreting the extracellular vesicles of claim 4, (A) a nucleic acid encoding a fusion protein comprising the first antibody of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) A nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) A nucleic acid encoding a fusion protein of an antigen peptide that binds to the first antibody of (c) and a protein present in extracellular vesicles. Extracellular vesicle-secreting cells, including

23. The extracellular vesicle-secreting cell of any one of claims 20 to 22, further comprising a nucleic acid encoding (D) the viral fusion protein (d).

24. (C) The fusion protein with the protein present in the extracellular vesicle of (c) contains a peptide corresponding to the barcode sequence and is presented outside the vesicle. The extracellular vesicle-secreting cell according to any one of claims 20 to 22.

25. The extracellular vesicle-secreting cell according to any one of claims 20 to 22, further comprising (E) a nucleic acid that affects the properties of extracellular vesicles, or an expression vector that expresses a nucleic acid that affects the properties of extracellular vesicles.

26. The nucleic acid that affects the properties of the extracellular vesicles (1) A nucleic acid that changes the amount of an endogenous protein present in or on the surface of an extracellular vesicle; (2) a nucleic acid that promotes or inhibits the secretion of extracellular vesicles; (3) nucleic acids that affect the lipid membrane that constitutes the membrane of extracellular vesicles; and (4) a nucleic acid for causing an exogenous protein to be present within or on the surface of the extracellular vesicle; The extracellular vesicle-secreting cell of claim 25.

27. The extracellular vesicle-secreting cell of claim 25, wherein the nucleic acid that affects the properties of the extracellular vesicle comprises mRNA, ncRNA, crRNA, or gRNA.

28. The extracellular vesicle-secreting cell of claim 27, further expressing Cas12a (Cpf1) endonuclease.

29. The extracellular vesicle-secreting cell according to any one of claims 20 to 22, wherein the extracellular vesicle-secreting cell is selected from the group consisting of HEK293 cells, stem cells, epithelial cells, endothelial cells, fibroblasts, cancer cells, immune cells, nerve cells, and plant cells.

30. A non-human organism comprising or consisting of a cell according to any one of claims 13 to 18.

31. 21. The cell of claim 20, wherein at least one nucleic acid selected from the group consisting of: (A) a nucleic acid encoding a fusion protein comprising the PrimeEditor enzyme containing the nuclear localization signal of (a); and (B) A nucleic acid encoding a PrimeEditing gRNA containing a complementary strand sequence of the barcode sequence in (b).

32. 22. The cell of claim 21, wherein the nucleic acid is at least one nucleic acid selected from the group consisting of: (A) a nucleic acid encoding a fusion protein comprising the first Tag peptide of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) A nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) A nucleic acid encoding a fusion protein of a tag peptide-binding protein that binds to a second tag peptide of (c) and the first tag peptide, wherein the binding affinity for the second tag peptide is greater than the binding affinity for the first tag peptide, and a protein present in extracellular vesicles.

33. 23. The cell of claim 22, wherein the nucleic acid is at least one nucleic acid selected from the group consisting of: (A) a nucleic acid encoding a fusion protein comprising the first antibody of (a) and a PrimeEditor enzyme comprising a nuclear localization signal; (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) A nucleic acid encoding a fusion protein of an antigen peptide that binds to the first antibody of (c) and a protein present in extracellular vesicles.

34. For producing the cell of claim 23, (D) A nucleic acid encoding the viral fusion protein of (d).

35. (C) The nucleic acid according to any one of claims 31 to 33, wherein the fusion protein with the protein present in the extracellular vesicle of (c) contains a peptide corresponding to the barcode sequence and is presented outside the vesicle.

36. The nucleic acid according to any one of claims 31 to 33, which is contained in an expression vector.

37. For producing the cell of claim 25, (E) A nucleic acid that affects the properties of extracellular vesicles, or an expression vector that expresses a nucleic acid that affects the properties of extracellular vesicles.

38. The nucleic acid that affects the properties of the extracellular vesicles (1) A nucleic acid that changes the amount of an endogenous protein present in or on the surface of an extracellular vesicle; (2) a nucleic acid that promotes or inhibits the secretion of extracellular vesicles; (3) nucleic acids that affect the lipid membrane that constitutes the membrane of extracellular vesicles; and (4) A nucleic acid for causing an exogenous protein to be present within or on the surface of an extracellular vesicle.

39. 38. The nucleic acid or expression vector of claim 37, wherein the nucleic acid that affects the properties of the extracellular vesicles comprises mRNA, ncRNA, crRNA, or gRNA.

40. For producing the cell of claim 28, Expression vector for expressing Cas12a (Cpf1) endonuclease.

41. For producing the cell of claim 13, (i) A nucleic acid encoding a target sequence for a PrimeEditing gRNA.

42. 15. The cell of claim 14, wherein at least one nucleic acid selected from the group consisting of: (i) a nucleic acid encoding a target sequence for the PrimeEditing gRNA; and (ii) A nucleic acid encoding a fusion protein comprising a second Tag peptide.

43. 43. The nucleic acid of claim 41 or 42, further comprising a sequence encoding a fluorescent protein and / or a label protein, wherein the barcode sequence is configured such that the fluorescent protein or label protein is transcribed and translated only when the barcode sequence is inserted into a target sequence.

44. 43. The nucleic acid of claim 41 or 42, contained in an expression vector.

45. A method for producing a library of extracellular vesicles, comprising: 1) Extracellular vesicle-secreting cells (i) a nucleic acid encoding a fusion protein containing a PrimeEditor enzyme containing a nuclear localization signal or an expression vector for expressing the fusion protein; and (ii) Multiple types a1) a nucleic acid comprising a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence of at least one barcode sequence; and a2) a nucleic acid comprising at least one sequence that affects the properties of extracellular vesicles corresponding to the barcode sequence. Combination of or, Multiple species a) a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence of at least one barcode sequence; and a nucleic acid encoding at least one sequence that affects the properties of extracellular vesicles corresponding to the barcode sequence. introducing 2) culturing the extracellular vesicle-secreting cells after the introduction in a culture medium; and 3) recovering extracellular vesicles containing the fusion protein containing the PrimeEditor enzyme and the PrimeEditing gRNA from the culture supernatant of extracellular vesicle-secreting cells; method.

46. The method of claim 45, wherein the extracellular vesicle-secreting cells express Cas12a (Cpf1) endonuclease, and the sequence that affects the properties of the extracellular vesicles is a gRNA or sgRNA sequence for an endogenous gene.

47. 1. A method for producing a library of extracellular vesicles that display peptides on their outer surface, comprising: 1) Extracellular vesicle-secreting cells (i) a nucleic acid encoding a fusion protein containing a PrimeEditor enzyme containing a nuclear localization signal or an expression vector for expressing the fusion protein; and (ii) Multiple types a) a nucleic acid comprising a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence of at least one barcode sequence; and b) a nucleic acid comprising a sequence encoding a fusion protein comprising a peptide corresponding to the barcode sequence and a protein present in extracellular vesicles for extravesicle presentation. Combination of or, Multiple species a1) a sequence encoding a PrimeEditing gRNA containing a complementary strand sequence of at least one barcode sequence; and a2) a sequence encoding a fusion protein containing a peptide corresponding to the barcode sequence and a protein present in extracellular vesicles that are presented outside the vesicles. Nucleic acid containing introducing 2) culturing the extracellular vesicle-secreting cells after the introduction in a culture medium; and 3) recovering extracellular vesicles that contain the fusion protein containing the PrimeEditor enzyme and the PrimeEditing gRNA and present the peptide from the culture supernatant of extracellular vesicle-secreting cells; method.

48. Before step 2 introducing a nucleic acid containing a sequence encoding a fusion protein of a tag peptide-binding protein that binds to a second tag peptide and the first tag peptide, the fusion protein having a higher binding affinity to the second tag peptide than to the first tag peptide, and a protein present in extracellular vesicles; (i) A fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal contains a first Tag peptide; 48. The method according to any one of claims 45 to 47.

49. Before step 2 introducing a nucleic acid containing a sequence encoding a fusion protein of a tag peptide-binding protein that binds to a second tag peptide and the first tag peptide, the fusion protein having a higher binding affinity to the second tag peptide than to the first tag peptide, and a protein present in extracellular vesicles; (i) A fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal contains a first Tag peptide; 48. The method according to any one of claims 45 to 47.

50. The first Tag peptide is AlfaTag ＰＥ (SEQ ID NO: 21), and the second Tag peptide is AlfaTag ＳＴ (SEQ ID NO: 22) and the Tag-binding protein comprises an anti-Alfa Nanobody (SEQ ID NO: 27).

51. Before step 2 introducing a nucleic acid containing a sequence encoding a fusion protein of an antigen peptide that binds to a first antibody and a protein present in extracellular vesicles; (i) A fusion protein comprising the PrimeEditor enzyme containing a nuclear localization signal comprises a first antibody; 48. The method according to any one of claims 45 to 47.

52. 52. The method of claim 51, wherein the antigen peptide is SUNtag peptide (GCN peptide x10) (SEQ ID NO: 24) and the first antibody is an anti-GCN antibody (SEQ ID NO: 25).

53. Before step 2 The method of any one of claims 45 to 47, further comprising the step of (d) introducing a nucleic acid comprising a sequence encoding a viral fusion protein.

54. The method of any one of claims 45 to 47, wherein the protein present in the extracellular vesicles comprises CD9 (SEQ ID NO: 28) or BASP1 (1-30) (SEQ ID NO: 29).

55. The method of any one of claims 45 to 47, wherein the barcode sequence comprises any nucleic acid sequence of 5 to 8 bases.

56. The barcode sequence is 56. The method of claim 55, wherein the amino acid sequence is 5'NNNNNNTGNN3' (wherein N is any base).

57. The method of any one of claims 45 to 47, wherein the PrimeEditing gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 13-20 and 63: 【Table 14】

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