Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
  • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof

Definitions

• the present invention relates to a system for recording the fate of extracellular vesicles.
• vesicles extracellular vesicles (EVs) secreted from cells, depending on their origin and characteristics.
• EVs extracellular vesicles
• the former are membrane vesicles with a diameter of about 30 to 200 nm, and contain many exosomes whose membranes are thought to be mainly derived from endosomes.
• MVs microvesicles
• ectosomes whose main components are thought to be cell membrane components, and apoptotic bodies formed by cell fragmentation during apoptosis
• MVs are formed by being directly constricted from the cell membrane, and are small vesicles with a size of approximately 200 nm to 1000 nm. MVs contain integrins, selectins, CD40, and the like. Apoptotic bodies are also formed by being directly constricted from the cell membrane, but are small vesicles measuring 500 nm to 2000 nm in size, and contain fragmented genomic DNA, histone proteins, and the like.
• Exosomes have been reported to be involved in close or long-distance cell-cell communication. For example, in the immune system, exosomes released from cells function as antigen-presenting vesicles and induce antitumor immune responses and immune tolerance that suppresses inflammation (Non-Patent Document 4). In neurodegenerative diseases, it is known that pathogenic proteins such as prions and beta-amyloid peptides use exosomes to spread to other cells.
• Exosomes have low immunogenicity and are capable of penetrating the blood-brain barrier.
• exosomes contain various nucleic acids (mRNA, miRNA, shRNA, ncRNA, etc.), and it is known that these nucleic acids function in exosome-receiving cells and affect the function of the exosome-receiving cells. Therefore, in recent years, there has been active research into using exosomes as drug delivery systems (DDS) in hopes of achieving therapeutic effects through functional modification of exosome-receiving cells (Non-Patent Document 5).
• DDS drug delivery systems
• Non-Patent Documents 6 to 9, Patent Document 1 attempts have been made to improve the efficiency of DDS by expressing peptides or proteins recognized by exosome-receiving cells on the membrane surface of exosomes to increase the efficiency of targeting to exosome-receiving cells, or by expressing RNA-binding proteins on the inner membrane side of exosomes to efficiently recruit RNA in the cytoplasm.
• Non-Patent Document 10 The present inventors have also developed exosomes suitable for DDS.
• Non-Patent Documents 11 and 12 These papers suggest that there is a relationship between the type of integrin present on the surface of exosomes and the type of organ to which cancer will metastasize.
• Extracellular vesicles are not only unique to animals, but are also present 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).
• Patent Document 2 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.
• 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.
• the inventors of the present application have succeeded in incorporating a fusion protein containing a PrimeEditing gRNA containing a complementary sequence to a barcode sequence and a PrimeEditor enzyme containing a nuclear localization signal into an extracellular vesicle.
• a receptor cell that has taken up the extracellular vesicle contains a nucleic acid in its nucleus that encodes a target sequence for the PrimeEditing gRNA
• the barcode sequence is inserted into the target sequence by a genome editing reaction.
• the present invention includes the following embodiments: (Extracellular vesicles) [A1] (a) a fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal; and (b) a PrimeEditing gRNA containing a complementary strand sequence of a barcode sequence. , extracellular vesicles.
• [A2] (a) a fusion protein comprising a PrimeEditor enzyme comprising a first Tag peptide and a nuclear localization signal; (b) a Prime Editing gRNA comprising a complementary strand sequence of a barcode sequence; and (c) a fusion protein of a tag peptide-binding protein that binds to a second tag peptide and the first tag peptide, the protein having a greater binding affinity for the second tag peptide than for the first tag peptide, and a protein present in the extracellular vesicle.
• [A3] The extracellular vesicle described in [A2], wherein the first tag peptide comprises AlfaTagPE (sequence number 21), the second tag peptide comprises AlfaTagST (sequence number 22), and the tag binding protein comprises an anti-Alfa nanobody (sequence number 27).
• [A4] (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 antigen peptide that binds to the first antibody and a protein present in the extracellular vesicle.
• [A5] The extracellular vesicle described in [A4], wherein the antigen peptide is a SUNtag peptide (GNC peptide x10) (sequence number 24) and the first antibody is an anti-GCN single chain antibody (sequence number 25).
• [A6] The extracellular vesicle described in any one of [A1] to [A5], further comprising (d) a viral fusion protein on its surface.
• [A7] (d) An extracellular vesicle described in [A6], wherein 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 contains a peptide corresponding to a 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.
• the barcode sequence is The extracellular vesicle described in [A9], which is 5'NNNNNNTGNN3' (N is any base).
• (Receiver cells) [B1] (i) a cell comprising a nucleic acid encoding a target sequence for a PrimeEditing gRNA in its nucleus; preferably wherein, when the extracellular vesicle described in [A1] is taken up into the 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 barcode sequence (b) is transported into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction.
• [B2] (i) a cell that contains a nucleic acid encoding a target sequence for the PrimeEditing gRNA in the nucleus; and (ii) a cell that contains a fusion protein containing the second Tag peptide in the cytoplasm; preferably, when the extracellular vesicle described in [A2] is 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 antagonistic action with the (ii) fusion protein comprising the second Tag peptide, and as a result, the (a) fusion protein comprising the first Tag peptide and the PrimeEditor enzyme comprising a nuclear localization signal, which encapsulates the PrimeEditing gRNA comprising the complementary strand sequence of the barcode sequence, is translocated into the nucleus, and the barcode sequence is inserted into the target sequence by
• [B3] The cell described in [B2], wherein the first tag peptide comprises AlfaTag PE (sequence number 21), the second tag peptide comprises AlfaTag ST (sequence number 22), and the tag binding protein comprises an anti-Alfa nanobody (sequence number 27).
• [B4] (i) A cell comprising a nucleic acid encoding a target sequence for a PrimeEditing gRNA in its nucleus; preferably wherein, when the extracellular vesicle described in [A4] is taken up into the cell, The adhesion between the antigen peptide (c) and the first antibody (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.
• the target sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 6: A cell described in 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.
• 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, neuronal cells, and plant cells.
• Donor cells [C1] A cell that secretes extracellular vesicles of [A1], (A) A nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme comprising 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).
• [C2] A cell that secretes extracellular vesicles of [A2], (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 Prime Editing 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, the binding affinity of the tag peptide-binding protein to the second tag peptide being greater than the binding affinity of the tag peptide to the first tag peptide, and a protein present in the extracellular vesicle.
• [C3] A cell that secretes extracellular vesicles of [A4], (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 Prime Editing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) an extracellular vesicle-secreting cell comprising 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 vesicle.
• [C4] The extracellular vesicle-secreting cell of any one of [C1] to [C3], further comprising a nucleic acid encoding a viral fusion protein (D) (d).
• [C5] (C) The extracellular vesicle-secreting cell according to any one of [C1] to [C4], 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.
• [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.
• the nucleic acid that affects the properties of the extracellular vesicle is (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) a nucleic acid that affects the lipid membrane that constitutes the membrane of the extracellular vesicle; 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 according to [C6], [C8] The extracellular vesicle secreting cell of claim C6, wherein the nucleic acid that affects the properties of the extracellular vesicle comprises mRNA, ncRNA, crRNA or gRNA. [C9] Further, the extracellular vesicle secreting cell described in [C8] expresses 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, neuronal cells and plant cells.
• [D1] A non-human organism comprising or consisting of a cell according to any one of [B1] to [B7].
• nucleic acid for producing donor cells [E1] [C1] For producing the cell, at least one nucleic acid selected from the following group: (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).
• nucleic acid For producing the cell, at least one nucleic acid selected from the following group: (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 Prime Editing 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, the binding affinity of the tag peptide-binding protein being greater than the binding affinity of the second tag peptide to the first tag peptide, and a protein present in extracellular vesicles.
• 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
• [E3] For producing the cell, at least one nucleic acid selected from the following group: (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 Prime Editing 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] To prepare cells of [C4], (D) A nucleic acid encoding the viral fusion protein of (d).
• the nucleic acid that affects the properties of the extracellular vesicle is (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) a nucleic acid that affects the lipid membrane that constitutes the membrane of an extracellular vesicle; and (4) a nucleic acid for causing an exogenous protein to be present within or on the surface of an extracellular vesicle.
• the nucleic acid or expression vector described in [E7] is selected from the group consisting of: [E9]
• the nucleic acid or expression vector described in [E7], wherein the nucleic acid that affects the properties of the extracellular vesicles includes mRNA or ncRNA (including miRNA, siRNA, shRNA, snRNA, snoRNA, gRNA, and sgRNA).
• mRNA or ncRNA including miRNA, siRNA, shRNA, snRNA, snoRNA, gRNA, and sgRNA.
• nucleic acid for producing receiver cells [F1] For producing cells of [B1] or [B4], (i) A nucleic acid encoding a target sequence for a PrimeEditing gRNA. [F2] [B2] For producing the cell, at least one nucleic acid selected from the following group: (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.
• [F3] The nucleic acid described in [F1] or [F2], further comprising a sequence encoding a fluorescent protein and/or a labeling protein, wherein the fluorescent protein or labeling protein is transcribed and translated only when the barcode sequence is inserted into a target sequence.
• [F4] The nucleic acid according to any one of [F1] to [F3], which is contained in an expression vector.
• a method for producing a library of extracellular vesicles comprising: 1) A step of 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 encoding 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)
• a method for producing a library of extracellular vesicles that display peptides on their outer surface comprising: 1) A combination of 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 in an extracellular vesicle-secreting cell, and (ii) a plurality of types of a) a nucleic acid containing a sequence encoding a PrimeEditing gRNA containing a complementary strand sequence of at least one barcode sequence; and b) a nucleic acid containing a sequence encoding a fusion protein containing a peptide corresponding to the barcode sequence and a protein present in the extracellular vesicles to be presented outside the vesicles, or A step of introducing a nucleic acid comprising: a1) a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence of at least one barcode sequence; and a2)
• the method includes the step of introducing a nucleic acid including 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 greater binding affinity to the second tag peptide than to the first tag peptide, and a protein present in the extracellular vesicle; (i) A fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal contains a first Tag peptide; The method according to any one of [G1] to [G3].
• the method includes the step of introducing a nucleic acid including 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 greater binding affinity to the second tag peptide than to the first tag peptide, and a protein present in the extracellular vesicle; (i) A fusion protein comprising a PrimeEditor enzyme containing a nuclear localization signal contains a first Tag peptide; The method according to any one of [G1] to [G3].
• the method includes a step of introducing a nucleic acid including 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 a PrimeEditor enzyme containing a nuclear localization signal comprises a first antibody; The method according to any one of [G1] to [G3].
• [G8] The method according to [G7], wherein the antigen peptide is a SUNtag peptide (GNC peptide x10) (sequence number 24) and the first antibody is an anti-GCN antibody (sequence number 25).
• the antigen peptide is a SUNtag peptide (GNC peptide x10) (sequence number 24) and the first antibody is an anti-GCN antibody (sequence number 25).
• step 2 The method according to any one of [G1] to [G8], further comprising the step (d) of introducing a nucleic acid comprising a sequence encoding a viral fusion protein.
• the protein present in the extracellular vesicles comprises CD9 (sequence number 28) or BASP1 (1-30) (sequence number 29).
• This invention can be useful for the development of efficient drug delivery systems using extracellular vesicles, for biology research of extracellular vesicles, and for drug discovery research targeting the extracellular vesicle secretion pathway.
• FIG. 1 shows the structure of a sequence including a target sequence for a receiver cell according to one embodiment of the present invention. By inserting a target sequence into the target sequence, GFP and luciferase are normally expressed.
• Figure 1B shows the efficiency of prime editing using pegRNAs #1 to #6 corresponding to each target sequence #1 to #6 in Example 1.
• FIG. 2A shows a schematic diagram of the sequence of pegRNA.
• Figure 2B shows the efficiency of Prime Editing using different pegRNA sequences in Example 2.
• FIG. 3A is a schematic diagram of an extracellular vesicle according to one embodiment of the present invention, and a schematic diagram of sequence information for producing the composition.
• FIG. 3B shows the amount of pegRNA in extracellular vesicles in Example 3.
• FIG. 3A is a schematic diagram of an extracellular vesicle according to one embodiment of the present invention, and a schematic diagram of sequence information for producing the composition.
• FIG. 3B shows the amount of pegRNA in extracellular ve
• FIG. 4A is a schematic diagram of an extracellular vesicle fate recording system according to one embodiment of the present invention, and a schematic diagram of sequence information for constructing the system.
• FIG. 4B shows the editing efficiency in Example 4 (flow cytometry).
• Figure 4C shows the editing efficiency in Example 4 (PCR:restriction enzyme).
• FIG. 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).
• the present invention includes an extracellular vesicle comprising: (a) a fusion protein comprising a PrimeEditor enzyme that includes a nuclear localization signal; and (b) a PrimeEditing gRNA that includes a complementary strand sequence of a barcode sequence.
• Extracellular (secretory) vesicles are vesicles used to release intracellular substances to the outside of the cell, and are formed by a bilayer of phospholipids.
• lipid compositions include sphingomyelin and phosphatidylserine.
• the size of EVs is 10 nm to 10 ⁇ m, 30 nm to 5000 nm, or 50 nm to 3000 nm in diameter, and 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 suitable for the present invention, but are not limited thereto.
• the origin of EVs 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 intestinal bacteria) are preferred.
• the PrimeEditor enzyme containing a nuclear localization signal is a fusion protein containing i) a nucleic acid programmable DNA binding protein (napDNAbp) used in PrimeEditing 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.
• Cas9 having a RuvC domain and an HNH domain
• it is preferable that the activity of the HNH domain is deleted and that the activity of introducing a nick into a single strand (i.e., nickase activity) is present.
• Cas9 may have nickase activity by having an amino acid substitution of H840A.
• Such Cas9 may have a natural amino acid sequence other than H840A as is, and may further include amino acid substitutions, deletions and additions as long as the activity is maintained.
• the polypeptide having RNA-dependent DNA polymerase activity may be, but is not limited to, a reverse transcriptase (RT) (EC 2.7.7.49).
• the reverse transcriptase is from the genus Lentivirus (including HIV, SIV, FIV, EIA), the genus Alpharetrovirus (including Avian Myeloblastosis Virus (AMV)), the genus Betaretrovirus, or the genus Gammaretrovirus (including Moloney Murine Leukemia Virus (MMLV)).
• a reverse transcriptase may have the natural amino acid sequence as is, or may contain amino acid substitutions, deletions, and additions as long as the activity is maintained.
• the napDNAbp and (ii) the polypeptide having RNA-dependent DNA polymerase activity may be fused via a linker.
• the nuclear localization signal also called 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 consisting of positively charged amino acids (lysine, arginine). Such a sequence may be contained in the PrimeEditor enzyme and serve as a marker for transporting the protein to the cell nucleus, and may be located anywhere on the PrimeEditor enzyme sequence ( NH2- terminus; COOH-terminus; or inside the enzyme). In one embodiment, a PrimeEditor enzyme containing such a nuclear localization signal may comprise the amino acid sequence of PE2 (SEQ ID NO:61), PE2* (SEQ ID NO:62) or PEmax (SEQ ID NO:30).
• a 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 (containing a complementary strand sequence of a barcode sequence), and a primer binding site in this order.
• the barcode sequence is a sequence containing any of 5, 6, 7, or 8 bases. It may contain 0 to 4 bases of a given base sequence, and may be 5 to 12 bases in length in total.
• PrimeEditing gRNA containing a complementary strand sequence of the barcode sequence can be bound to the PrimeEditor enzyme and inserted into the target sequence by the Prime Editing reaction.
• the barcode sequence may be inserted in a form in which a sequence of 1, 2, 3, 4, 5, 6, 7 or 8 consecutive bases in the target sequence is replaced with the barcode sequence.
• the combination of the pegRNA sequence and the following target sequence can be arbitrarily set based on the descriptions in Non-Patent Documents 18 and 19.
• the fusion protein containing the 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 vesicle may further contain (c) a fusion protein of a Tag peptide binding protein that is a protein that binds to the second Tag peptide and the first Tag peptide and has a greater binding affinity to the second Tag peptide than the binding affinity to the first Tag peptide, and a protein present in the extracellular vesicle.
• the PrimeEditor enzyme (a) encapsulating the PrimeEditing gRNA (b) is linked to a protein present in the extracellular vesicle through binding with the first Tag peptide and the protein that binds to the Tag peptide, thereby enabling it to be localized by the extracellular vesicle.
• the fusion protein containing the PrimeEditor enzyme containing a nuclear localization signal may contain a first antibody or may be fused with the first antibody, and the extracellular vesicle may further contain (c) a fusion protein of an antigen peptide that binds to the first antibody and a protein present in the extracellular vesicle.
• the PrimeEditor enzyme (a) encapsulating the PrimeEditing gRNA (b) is linked to a protein present in the extracellular vesicle through the bond between the first antibody and the antigen peptide, thereby enabling it to be localized by the extracellular vesicle.
• the protein present in the extracellular vesicles is preferably an extracellular vesicle marker, i.e., a protein whose detection proves the presence of (specific) extracellular vesicles (i.e., a protein that is abundantly present in extracellular vesicles or that is specifically present in extracellular vesicles).
• extracellular vesicle marker i.e., a protein whose detection proves the presence of (specific) extracellular vesicles (i.e., a protein that is abundantly present in extracellular vesicles or that is specifically present in extracellular vesicles).
• extracellular vesicle marker i.e., a protein whose detection proves the presence of (specific) extracellular vesicles (i.e., a protein that is abundantly present in extracellular vesicles or that is specifically present in extracellular vesicles).
• non-human mammals including mice and rats
• higher plants or microorganisms
• 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) Non-tissue specific: Tetraspanins (CD63, CD9, CD81, CD82), other multi-transmembrane 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 syndecans (SDC)); Extracellular matrix metalloproteinase inducer (EMMPRIN) (also called BSG or CD147); ADAM10; CD73 (NT5E), a GPI-anchored 5'nucleotidase; GPI-anchored complement-binding proteins CD55 and
• 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); E.H.D.; RHOA; Annexin (ANXA); the heat shock proteins HSC70 (HSPA8) and HSP84 (HSP90AB1); ARF6; Syntenin (SDCBP); Microtubule-associated protein tau (Tau, MAPT; neuron-specific) etc.
• ALIX PDCD6IP
• VPS4A/B VPS4A/B
• ARRDC1 Flotillin-1 and 2
• FLOT1/2 Flotillin-1 and 2
• Caveolin CAV
• E.H.D. RHOA
• Annexin ANXA
• HSPA8 and HSP84 HSP84
• SDCBP Synten
• LAMP2B 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.
• the protein present in the extracellular vesicles may be a naturally occurring protein (including polymorphism, orthologs, and paralogs). Alternatively, it may be an artificial mutant in which some amino acids have been added, substituted, or deleted, or a fragment thereof (for example, "exoTOPE” or 1 to 30 amino acid residues of BASP1), but an artificial mutant or fragment that does not change the localization of the protein is preferred.
• 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 a cell. More preferably, the binding affinity of the Tag binding protein to the second Tag peptide is greater than the binding affinity of the Tag binding protein to the first Tag peptide.
• such combinations may include any peptide (5 to 20 amino acid residues) as a first tag peptide; a single chain antibody or a binding active fragment thereof against the peptide as a 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 a second tag peptide.
• such a combination is preferably the AlfaTag system described in Patent Document 6,
• the first tag peptide and the second tag peptide may comprise an amino acid sequence set forth in any of SEQ ID NOs: 64 to 177, and although not particularly limited thereto, it is preferable that the first tag peptide comprises AlfaTag PE (SEQ ID NO: 21) and the second tag peptide comprises 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).
• 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.
• a GNC peptide SEQ ID NO: 23
• SunTag SEQ ID NO: 24
• an anti-GCN antibody SEQ ID NO: 25
• the antigen peptide is capable of binding to and dissociating from the first antibody within a cell.
• a combination may use any peptide (5 to 20 amino acid residues) as an antigen peptide; and a single chain antibody against the peptide or a binding active fragment thereof as the first antibody.
• such combinations include a combination of AlfaTag of the AlfaTag system with an AlfaTag antibody; or a combination of SunTag with a SunTag antibody.
• the extracellular vesicles contain a viral fusion protein or a fusion-active fragment thereof on their surface.
• the fusion proteins of such enveloped viruses can be classified into three types, Class I to Class III, based on their structural features: Class I fusion proteins: influenza virus hemagglutinin (HA) and retrovirus Env, etc.; Class II fusion proteins: fusion proteins of Semliki Forest Virus (SFV) and Dengue virus, etc.; Class III fusion proteins: The G protein (VSV-G) of the rhabdovirus Vesicular Stomatitis Virus (VSV) and gB of herpes viruses.
• the viral fusion protein preferably comprises VSG-G or a fusion active fragment thereof.
• VSG-G may comprise the amino acid sequence of SEQ ID NO: 26, and includes 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.
• 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 the activity is maintained.
• a viral fusion protein or a fusogenic fragment thereof on the surface of the extracellular vesicle promotes fusion of the extracellular vesicle with the cell membrane of the receptor cell, facilitating the uptake of the extracellular vesicle (containing the fusion protein containing the PrimeEditor enzyme and the PrimeEditing gRNA) into the cell.
• the barcode sequence is 5'NNNNNNTGNN3', where N is any base.
• (b) a PrimeEditing gRNA comprising a complementary strand sequence of a barcode sequence The sequence includes a sequence selected from the group consisting of SEQ ID NOs: 13-20 and 63:
• the present invention includes receiver cells that take up the extracellular vesicles and record the barcode sequence.
• a receiver cell is a cell that (i) comprises in its nucleus a nucleic acid encoding a target sequence for the PrimeEditing gRNA; preferably wherein, when the extracellular vesicles are taken up into the cell, (b) 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 is transported into the nucleus, and the barcode sequence is inserted into the target sequence by a genome editing reaction.
• such a receiver cell contains a nucleic acid encoding a target sequence for a PrimeEditing gRNA in the nucleus; wherein, when the extracellular vesicle is taken up into the cell, the adhesion between the Tag binding protein of (c) and the first Tag peptide 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 a barcode sequence (b) is 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 the cytoplasm, wherein the adhesion between the (c) Tag peptide-binding protein and the (a) first Tag peptide is dissociated by the antagonistic action with the fusion protein comprising the second Tag peptid
• such a receiver cell contains a nucleic acid encoding a target sequence for the PrimeEditing gRNA in the nucleus; wherein the adhesion between the antigen peptide of (c) and the first antibody of (a) is dissociated, and as a result, (b) a fusion protein containing a PrimeEditor enzyme including a nuclear localization signal, which harbors 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; More preferably, the cell contains (ii) a fusion protein comprising the first antibody in the cytoplasm, wherein adhesion between the antigen peptide of (c) and the first antibody of (a) is dissociated by antagonistic action with the fusion protein comprising the first antibody of (ii), and as a result, (b) the fusion protein comprising the first Tag peptide (a) and the PrimeEdit
• the target sequence may be a sequence that is native to the receiver cell or may be an exogenous sequence. Such a sequence is preferably present in the cell (in the cytoplasm, nucleus, or genome) in the form of DNA and is not transient.
• the target sequence includes the sequence of 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.
• the receiver cell includes 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.
• 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.
• the sequence information is configured so that the mRNA encoding the fluorescent protein and/or labeled protein is transcribed only when the barcode sequence is inserted into the target sequence.
• the receiver cell may be a HEK293 cell, a stem cell, an epithelial cell, an endothelial cell, a fibroblast cell, a cancer cell, an immune cell, a neuronal cell or a plant cell.
• the present invention includes donor cells that secrete the above-mentioned extracellular vesicles.
• a donor cell comprises (A) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme comprising 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).
• the cell transcribes and translates the fusion protein of (a) and transcribes the PrimeEditing gRNA of (b).
• the transcribed and translated fusion protein of (a) and the PrimeEditing gRNA of (b) form a complex within the cell, are incorporated into extracellular vesicles, and are secreted outside the cell.
• such a donor cell comprises (A) a nucleic acid encoding a fusion protein comprising a first Tag peptide and a PrimeEditor enzyme comprising a nuclear localization signal of (a); (B) a nucleic acid encoding a PrimeEditing gRNA comprising a complementary strand sequence of the barcode sequence of (b); and (C) a protein that binds to the second Tag peptide of (c) and the first Tag peptide, the binding affinity for the second Tag peptide being greater than the binding affinity for the first Tag peptide, and a nucleic acid encoding a fusion protein of a protein present in an extracellular vesicle.
• 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 of (a) and the PrimeEditing gRNA of (b) form a complex within the cell, and are further linked to the fusion protein of (c), thereby being specifically incorporated into extracellular vesicles and secreted outside the cell.
• such donor cells comprise (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.
• 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 of (a) and the PrimeEditing gRNA of (b) form a complex within the cell, and are further linked to the fusion protein of (c), thereby being specifically incorporated into extracellular vesicles and secreted outside the cell.
• the donor cell further comprises a nucleic acid encoding a viral fusion protein (D) of (d).
• the cell transcribes and translates the viral fusion protein (d), and the transcribed and translated viral fusion protein (d) is presented on the outer surface of the secreted extracellular vesicles, thereby promoting adhesion between the extracellular vesicles and the receiver cells.
• a fusion protein with a protein present in the extracellular vesicle of (C) (c) contains a peptide corresponding to the barcode sequence and is presented outside the vesicle.
• the barcode sequence and the peptide do not have to have a one-to-one relationship, and may have a one-to-multiple type, that is, multiple types of peptides may be linked to one type of barcode sequence.
• such donor cells contain nucleic acids that affect the properties of the extracellular vesicles.
• a nucleic acid that affects the properties of extracellular vesicles refers to a nucleic acid that, when a cell secreting extracellular vesicles contains the nucleic acid, by analyzing the extracellular vesicles secreted from the cell, (1) the amount of endogenous protein present in or on the surface of the secreted extracellular vesicles is changed compared to when the cell secreting extracellular vesicles does not contain the nucleic acid; (2) the amount of extracellular vesicles secreted is changed compared to when the cells secreting the extracellular vesicles do not contain the nucleic acid; (3) affecting the lipid membrane that constitutes the extracellular vesicle membrane compared to when the cell secreting the extracellular vesicles does not contain the nucleic acid; (4) the exogenous protein encoded by the nucleic acid is present within or on the surface of the extracellular vesicle;
• the nucleic acid may be naturally occurring DNA or RNA
• nucleic acid may also be a non-naturally occurring nucleic acid (for example, a nucleic acid in which the nucleotides are not bound by phosphorylated esters (P) but are partially or entirely S-substituted (phosphorothioate), or a peptide nucleic acid (PNA), etc.).
• P phosphorylated esters
• PNA peptide nucleic acid
• Examples of (1) include nucleic acids encoding endogenous proteins themselves, nucleic acids encoding transcription factors that control the transcription of endogenous proteins, nucleic acids encoding factors involved in the post-translational modification of endogenous proteins, and nucleic acids encoding factors involved in chaperoning (including folding and intracellular transport) of endogenous proteins; and nucleic acids encoding proteins such as antisense RNA (siRNA), miRNA (microRNA), shRNA (small hairpin RNA), and snRNA (small nuclear RNA) that positively or negatively control the expression of endogenous proteins, or nucleic acids for modifying genes encoding endogenous proteins using genome editing techniques (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.
• nucleic acids examples 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 per se; antisense RNA, miRNA, shRNA, and snRNA that positively or negatively control 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 per se, and nucleic acids for modifying genes encoding 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 per se in genome editing technology (e.g., ZFN, TALEN, CRISPR/Cas9) (e.g., gRNA used in the CRISPR/Cas9 system).
• genome editing technology e.g
• (3) includes nucleic acids encoding enzymes for synthesizing the lipid membrane constituting the extracellular vesicle membrane, nucleic acids encoding factors for controlling the transcription, translation and expression of enzymes for synthesizing the lipid membrane constituting 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 constituting the extracellular vesicle membrane and enzymes for synthesizing the lipid membrane constituting the extracellular vesicle membrane, or nucleic acids for modifying genes encoding factors for controlling the transcription, translation and expression of enzymes for synthesizing the lipid membrane constituting the extracellular vesicle membrane in genome editing technology (e.g., ZFN, TALEN, CRISPR / Cas9, etc.) (e.g., gRNA used in the CLISPR / Cas9 system).
• (4) includes nucle
• the nucleic acid that affects the properties of extracellular vesicles may include mRNA and ncRNA.
• mRNA refers to RNA including RNA (cRNA; coding RNA) that has 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 and RNA that does not contain polyadenylation (polyA) at the 3' end. It also includes premature mRNA that, if properly spliced within a cell, will have the base sequence information and structure to be translated into a protein.
• cRNA coding RNA
• Non-coding RNA refers to RNA (functional nucleic acid) that does not have 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) that form complexes with proteins in the nucleus, miRNA (including pre-miRNA) and siRNA (including pre-siRNA (e.g., shRNA (small hairpin RNA)) that bind to other RNA. Furthermore, the ncRNA may also include guide RNA (gRNA; including single-stranded guide RNA) (RNA that guides the RNA:protein complex to a target nucleic acid molecule by complementary binding).
• gRNA guide RNA
• CRISPR RNA CRISPR RNA
• tracrRNA trans-activating crRNA
• sgRNA single guide RNA
• nucleic acid-programmed DNA-binding protein different from the i) nucleic acid-programmed DNA-binding protein (napDNAbp) contained in the PrimeEditor enzyme.
• a nucleic acid-programmed DNA-binding protein different from the i) nucleic acid-programmed DNA-binding protein (napDNAbp) contained in the PrimeEditor enzyme.
• the i) nucleic acid-programmed DNA-binding protein (napDNAbp) contained in the PrimeEditor enzyme is Cas9
• 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 affects the properties of extracellular vesicles modify the genes of the donor cell, which in turn affects the properties of the extracellular vesicles secreted by the cell.
• the donor cells according to 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 may be 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, or may be cell lines of these cells.
• Cultured cells e.g., HEK293T cells
• nucleic acids for producing the donor cells described above.
• such a nucleic acid includes (A1) a nucleic acid encoding a fusion protein comprising a PrimeEditor enzyme containing 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) (c) a nucleic acid encoding a fusion protein between a tag peptide-binding protein that binds to a second tag peptide and the first tag peptide, the tag peptide-binding protein having a greater binding affinity to the
• A1-3, C1 and C2 are used to transcribe and translate the fusion protein, and B is used to transcribe the PrimeEditing 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 the donor cell.
• the nucleic acid includes a nucleic acid encoding (D) the viral fusion protein of (d). D is used to transcribe and translate the viral fusion protein.
• the nucleic acid may be linked to a suitable 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.
• such nucleic acids include (E) nucleic acids that affect the properties of extracellular vesicles.
• the nucleic acid may be linked to a promoter sequence suitable for such transcription and translation, or may be included in an expression vector for transcription and translation in the donor cell.
• the nucleic acid that affects the properties of the extracellular vesicles includes (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 the extracellular vesicle; and (4) Nucleic acids for causing an exogenous protein to be present within or on the surface of the extracellular vesicle, including mRNA or ncRNA (including miRNA, siRNA, shRNA, snRNA, snoRNA, gRNA, and sgRNA).
• mRNA or ncRNA including miRNA, siRNA, shRNA, snRNA, snoRNA, gRNA, and sgRNA.
• the nucleic acid includes an expression vector for expressing a nucleic acid programmable DNA binding protein (napDNAbp).
• napDNAbp nucleic acid programmable DNA binding protein
• the present invention includes nucleic acids for producing the above receiver cells.
• the nucleic acid comprises (i) a nucleic acid encoding a target sequence for a PrimeEditing gRNA; and/or (ii) a nucleic acid encoding a fusion protein that includes a second Tag peptide.
• the nucleic acid encoding the target sequence for the Prime Editing gRNA may further include a sequence encoding a fluorescent protein and/or a labeling protein, and may be configured such 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 comprised in an expression vector.
• the present invention includes a non-human organism that contains or consists of the receiver cell.
• the non-human organism may be a plant, an animal (including a mouse or a rat), or a microorganism. Such an organism may be produced by transplanting the receiver cell into an organism, or may be produced by a method known as a transgenic animal using a nucleic acid for producing the receiver cell.
• the present invention includes a method for producing a library of the above-mentioned extracellular vesicles.
• the method includes 1) administering to 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 step of introducing a nucleic acid encoding a plurality of 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 ves
• the extracellular vesicle-secreting cell expresses Cas12a (Cpf1) endonuclease, and the sequence that affects the properties of the extracellular vesicle may be a sequence of gRNA or sgRNA for an endogenous gene.
• the present invention includes a method for producing a library of extracellular vesicles that display peptides on their outer surface.
• the method includes 1) administering to 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 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 the extracellular vesicle for presentation outside the vesicle, or A step of introducing a nucleic acid comprising: a1) a sequence encoding a PrimeEditing gRNA comprising a complementary strand sequence of at least one
• the method includes a step of introducing a nucleic acid including 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 greater binding affinity to the second tag peptide than to the first tag peptide, and a protein present in the extracellular vesicle; (i) The fusion protein containing the PrimeEditor enzyme containing a nuclear localization signal may contain a first Tag peptide; or The method includes a step of introducing a nucleic acid including 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 greater binding affinity to the second tag peptide than to the first tag peptide, and a protein present in the extracellular vesicle; (i) The fusion protein comprising the PrimeEditor enzyme containing
• the method includes a step of introducing a nucleic acid including a sequence encoding a fusion protein of an antigen peptide that binds to a first antibody and a protein present in extracellular vesicles, (i)
• the fusion protein comprising the PrimeEditor enzyme containing a nuclear localization signal may comprise a first antibody;
• the antigen peptide may be a SUNtag peptide (GNC peptide x10) (SEQ ID NO: 24)
• the first antibody may be an anti-GCN antibody (SEQ ID NO: 25).
• the method may further comprise the step of (d) introducing a nucleic acid comprising a sequence encoding a viral fusion protein.
• 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 5-8 bases;
• the barcode sequence is 5'NNNNNNTGNN3' (where N is any base);
• the PrimeEditing gRNA is a sequence selected from the group consisting of SEQ ID NOs: 13-20 and 63: may include:
• the method for producing a library of extracellular vesicles includes at least two types of extracellular vesicles, and the identification of the extracellular vesicles is performed by determining the sequence of the barcode RNA encapsulated therein.
• the library preferably includes 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.
• a specific type of extracellular vesicles may be collected by using a specific extracellular vesicle marker as an indicator.
• the library of extracellular vesicles is added to the receiver cells, and the barcode sequences taken up by the receiver cells can be used to detect which extracellular vesicles have been taken up by the receiver cells.
• the library of extracellular vesicles can be administered to a non-human organism that contains/or consists of the receiver cells, and the barcode sequence incorporated into the receiver cells can be used to detect which extracellular vesicles have been incorporated into which receiver cells in which tissues.
• Primary screening to determine whether or not the extracellular vesicles have been taken up may be performed by detection using PCR or the like, or by treating the extracted DNA of the receiver cells with a restriction enzyme specific to the target sequence (the restriction enzyme recognition site is lost when the barcode sequence is inserted).
• the primary screening may be performed by detecting the fluorescent protein or labeling protein.
• Secondary screening to determine which extracellular vesicles have been taken up may be performed by sequencing the sequence of the target sequence region.
• the library of extracellular vesicles is (1) Identification of factors involved in changes in the amount of extracellular vesicles secreted (including factors involved in changes in the 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 that affect the half-life and dynamics of extracellular vesicles in body fluids; (4) Identification of factors that influence the targeting of extracellular vesicles to each tissue or body fluid; (5) Identification of factors that affect the targeting of extracellular vesicles to specific cells (including primary cells); It can be used in the following ways:
• Example 1 Selection of target sequence
• Construct for target sequence (mCherry-T2A-target-GFP-E2A-luciferase)
• Figure 1A A schematic diagram of the construct introduced into the receiver cell is shown in Figure 1A.
• the following #1 to #6 were used as target sequences.
• the target sequences were designed to contain the following BcgI or BsaXI restriction enzyme recognition sequences (restriction enzyme recognition sequences are shown in lower case).
• the sequence information of the construct in which #5 is 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 prepare an expression vector.
• PrimeEditing inserts and/or replaces barcode sequences targeting the underlined bases (inserting 5 bases before the underlined bases). Only when the barcode sequence is replaced, the fluorescent protein (GFP) and marker protein (luciferase) downstream of the target sequence are translated and expressed normally.
• GFP fluorescent protein
• marker protein luciferase
• PEGRNA Expression Constructs capable of transcribing the following PEGRNAs #1 to #6 were prepared in a form corresponding to the above target sequences #1 to #6.
• the vector plasmid for transcribing PEGRNA was prepared by introducing a 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).
• PEmax Expression Construct A sequence encoding PEmax (SEQ ID NO:30) was inserted into the SfiI site of pSBbi-Hyg (addgene #60524) to generate an expression vector.
• A) Each target sequence construct, 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 the expression of mCherry and GFP was analyzed using a flowcytometer. D-luciferin was also added to the cells, and the bioluminescence emitted from the cells was observed using a plate reader. The results are shown in Figure 1B. It shows that in all of the combinations of target sequences #1 to #6 and pegRNA used, the PrimeEdit reaction occurred in a pegRNA-dependent manner, and as a result, GFP and luciferase activities were detected.
• a stable cell line containing target sequence #5 was generated using sleeping beauty transposase with HEK293T cells as parent cells.
• the optimized pegRNA, Prime Editor (PEmax) was transiently transfected into the cell in the same manner as in Example 1. Two days after transfection, D-luciferin was added, and bioluminescence emitted from the cells was observed using a plate reader. The relative editing efficiency was calculated from the bioluminescence values and is shown in Figure 2B. PrimeEdit reaction occurred using all PEGRNAs.
• Example 3 Incorporation of pegRNA into extracellular secretory vesicles via the AlFaTag system and the SunTag system
• AlFaTag system and the SunTag system were introduced ( FIG. 3A ).
• A. Expression Construct 1. CD9-antiALFANb A vector plasmid expressing the CD9-anti-ALFA Nanobody fusion protein (SEQ ID NO: 40) was constructed by introducing a sequence encoding the CD9-anti-ALFA Nanobody fusion protein under 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 under the CMV promoter of pcDNA3.1(+). 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 under 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 under 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. antiGCNscFv-PE A vector plasmid expressing the 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) to prepare an expression vector. 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) to prepare an expression vector.
• pegRNA A vector plasmid for transcribing pegRNA (SEQ ID NO: 47) was prepared by introducing a sequence encoding pegRNA into pU6-pegRNA-GG-acceptor (addgene #132777).
• B. EV Production HEK293T cells cultured in 24 wells/plate were transfected with the above combinations of fusion protein expression vectors and pegRNA transcription vectors in the following combinations and ratios.
• Control cells Only the pegRNA expression plasmid (pSY62 AGGAA insertion) was transiently expressed.
• CD9-PE TSV cleavable
• CD9-PE expression plasmid and pegRNA expression plasmid were transiently expressed.
• SUNtag-system anchor (CD9-SUNtag or BASP1 (1-30)-SUNtag), a fusion of Prime Editor and anti-GCN antibody, and a pegRNA expression plasmid were transiently expressed.
• -ALFAtag system anchor (CD9-antiALFA nanobody or BASP1 (1-30)-SUNtag), fusion of Prime Editor and ALFA tag (ALFA(ST) here), and pegRNA expression plasmid were transiently expressed.
• anchor CD9-antiALFA nanobody or BASP1 (1-30)-SUNtag
• ALFA(ST) fusion of Prime Editor and ALFA tag
• pegRNA expression plasmid were transiently expressed.
• the medium was replaced, and 48 hr after the replacement, the supernatant was collected. After removing impurities from the supernatant using a filter, RNA was extracted from the supernatant, and the amount of pegRNA was quantified by qPCR.
• Figure 3B shows that pegRNA could be incorporated into EVs using both the AlFaTag and SunTag systems.
• Example 4 Effect of antagonistic Tag peptide and viral fusion protein on 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) promotes the fusion of extracellular vesicles with receiver cells, and the antagonistic Tag peptide (AlFaTag ST ) makes it easier for PE (+pegRNA) taken up into cells to dissociate from a protein (CD9) present in the extracellular vesicles, thereby promoting nuclear translocation (FIG. 4A).
• 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 under the EF-1 ⁇ promoter of the pSBbi-GH vector (addgene, plasmid #60514; hygromycin resistance gene + EGFP co-expression type). 2.
• AlfaTag PE -PE A vector plasmid expressing the AlfaTag PE -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.
• pegRNA Each vector plasmid for transcribing pegRNA (SEQ ID NO:63) (pRK455 (barcode: AGGAAT); pSY115 (barcode: CATTCA); pSY117 (barcode: GCCTAA), pSY120 (barcode: TGTCGA)) was prepared by introducing a sequence encoding pegRNA into pU6-tevopreq1-GG-acceptorp (addgene #174038). 4. VSV-G pMD2.G (Addgene #12259), which expresses VSV-G (SEQ ID NO:26), was utilized.
• AlFa ST mCherry-T2A-target-GFP-E2A-luciferase (SEQ ID NO: 49)
• SEQ ID NO: 49 AlFa ST mCherry-T2A-target-GFP-E2A-luciferase
• VSV-G VSV-G only, or the same amount of filler plasmid (pcDNA3.1) in the case of no VSV-G) were transfected into EV-producing cells.
• the medium was changed one day after transfection. Two days after medium change, the supernatant containing EVs was collected and contaminants were removed using a filter. D1.
• the filter-treated supernatant prepared in PrimeEdit reaction C1 was applied to HEK293T cells stably expressing the reporter prepared in B, and after 72 hours of culture, the cells were harvested and detached, and the fluorescence of mCherry and GFP was measured using a flowcytometer (FIG. 4B).
• VSV-G VSV-G alone, or without VSV-G, the same amount of filler plasmid (pcDNA3.1) was transfected into EV-producing cells. The medium was replaced one day after transfection. Two days after medium replacement, the supernatant containing EVs was collected and contaminants were removed using a filter.
• A) pRK455 (AGGAAT) or B) pSY107 (AGGACA) was used as an expression vector for pegRNA
• C) pSY115 (CATTCA), D) pSY117 (GCCTAA) or E) pSY120 (TGTCGA) was used as an expression vector for pegRNA.
• the filter-treated supernatant prepared in PrimeEdit reaction C2 was applied to HEK293T cells stably expressing the reporter prepared in B, and after 72 hours of culture, the cells were harvested, detached, and genomic DNA was extracted.
• the target sequence region was amplified by PCR using this as a template, treated with BsaXI, and the unedited target sequence was removed.
• the amount of barcode was then quantified by qPCR (FIG. 4C).
• Figures 4B and 4C show that the viral fusion protein (VSV-G) and the second Tag peptide (Alfa ST ) that competes with the first Tag peptide (Alfa PE ) promote the EV-mediated PrimeEdit reaction.
• Example 5 Insertion of a longer barcode sequence
• the length of the barcode sequence to be inserted into the target sequence was examined.
• one base on the target sequence for example, the underlined c in #5 target sequence: TgAATTAGCTACCTGTGAGAa -c -TGTGGctccGTTGGCGTCCGAAGCT
• TgAATTAGCTACCTGTGAGAa -c -TGTGGctccGTTGGCGTCCGAAGCT is inserted in a form that replaces the target sequence, but we verified whether the barcode sequence could be inserted in a form that includes several bases downstream of that base (Figure 5A).
• the vector plasmid for transcribing the pegRNA (SEQ ID NO: 50 to 60) that allows the following mixbase insertion sequence was prepared using addgene 174038 pU6-tevopreq1-GG acceptor as a backbone, encoding the pegRNA under the U6 promoter. #19 (SEQ ID NO: 37) used in Example 2 was used as a control.
• a stable cell line containing target sequence #5 was generated using sleeping beauty transposase with HEK293T cells as parent cells.
• the above pegRNA expression vector and Prime Editor (PEmax) expression vector were transiently transfected into the cell in the same manner as in Example 2. Two days after transfection, D-luciferin was added, and bioluminescence emitted from the cells was observed with a plate reader. The results are shown in Figure 5B. PrimeEdit reaction occurred using all of the pegRNAs.
• the 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 (e.g., elucidation of EV secretion in various cells), and in drug discovery research targeting the exosome secretion pathway (e.g., identification of factors that change the amount of EV secretion in a cell-specific manner).
• the present invention can also be used to analyze exosome-mediated networks across biological kingdoms.
• the mammalian digestive tract in addition to plants (food), intestinal bacteria, pathogenic microorganisms, and yeasts in food constantly form relationships that interact with each other, and the present invention can be useful in researching the exosomes produced by these heterogeneous biological communities.

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