Lipid bilayer membrane particles and method for producing same

Abstract

The combination is one or more combinations of a first engineered polypeptide comprising a multimerization sequence I and a cargo entity and a second engineered polypeptide comprising a multimerization sequence II that can associate with the multimerization sequence I and an I-BAR (Inverse-Bin / Amphiphysin / Rvs) domain. Also provided are engineered lipid bilayer membrane particles that include a first engineered polypeptide and a second engineered polypeptide.

Description

Lipid bilayer particles and method for producing the same 【0001】 This disclosure relates to lipid bilayer particles (LBPs) and methods for producing the same. 【0002】 The following explanation is provided solely for the purpose of helping readers understand this disclosure and does not constitute or describe prior art to this disclosure. 【0003】 Extracellular vesicles (EVs), such as exosomes and microvesicles, are nanometer-sized lipid bilayer particles produced by many types of cells that transport proteins, nucleic acids, and other substances between cells in the human body and other animals. EVs hold potential for a variety of therapeutic applications and are an attractive platform for delivering various therapeutic drugs. For example, targeted exosomes have already been shown to be effective in delivering RNA to nerve cells and tumor cells in mice. Other cell-derived membrane particles can be used for similar purposes. 【0004】 Extracellular vesicles are broadly classified into two types: vesicles derived from endosomes, which are part of the intracellular protein degradation pathway, and vesicles derived from the cell membrane, also known as ectosomes. Of these, endosomal vesicles are approximately 50-150 nm in size, and their secretion mechanisms have been studied in detail. A proposed secretion mechanism involves endosomes invaginating to form a polytope, which is then transported to the cell membrane where it fuses with the membrane, and the vesicles within are secreted (Non-Patent Literature 1). 【0005】Examples of modifying extracellular vesicles of the endosomal system have been reported (Non-Patent Literature 2). Examples of modifying extracellular vesicles of the cell membrane system by combining them with viral proteins to form virus-like particles have also been reported (Non-Patent Literature 3). Examples of modifying tetraspanins expressed in endosomes have also been reported (Non-Patent Literature 4). Furthermore, EVs designed with a loading system based on a pair of proteins that can be dimerized by abscisic acid have been reported to increase the likelihood that both membrane-bound anti-CD2 scFv will be incorporated into specific vesicles for the purpose of targeting cargo proteins and T cells (Non-Patent Literature 5). In these examples, the modified EVs are recovered into a small-EV fraction, and ultracentrifugation or prolonged centrifugation is used for their isolation. 【0006】 Cargo proteins can be loaded into EVs through overexpression in EV-producing cells, a method often referred to as "passive loading." 【0007】 Guillaume van N. et al., Nat. Rev. Mol. Cell Biol., 19(4), 213-228 (2018)Heath N. et al., Nanomedicine (Lond) 14(21), 2799-2814 (2019)Gee P et al., Nat Comm., 11, 1334 (2020)Zhang C. et al., Elife, 12:e84391 (2023)Stranford DM et al., Nat. Biomed. Eng., 8 (4), 397-414 (2024) 【0008】Passive loading is inefficient, especially for preparing membrane particles or extracellular vehicles (EVs) containing large cargo entities. In cases where extracellular vesicles of the cell membrane system are combined with viral proteins to create virus-like particles, there are also challenges related to immunogenicity posed by the viral-derived proteins. Furthermore, there is a need to manipulate multifunctional vesicles, such as EVs containing multiple concentrated cargo entities. The technology described herein aims to address these limitations of current technologies. Specifically, the technology aims to provide lipid bilayer particles containing high concentrations of any desired cargo entity and exhibiting low immunogenicity derived from exogenous viral proteins. 【0009】 This disclosure provides engineered polypeptides for loading cargo entities such as proteins and nucleic acids into lipid bilayer particles, particularly EVs, especially cell membrane-derived EVs (e.g., lipid bilayer particles including, but not limited to, extracellular vesicles (e.g., cell membrane-derived vesicles (CDMV), particularly filopodium-derived vesicles (FDV))), nucleic acids encoding such polynucleotides, vectors containing such nucleic acids, host cells containing such vectors, methods for loading such engineered polypeptides into lipid bilayer particles, methods for producing lipid bilayer particles containing such engineered polypeptides, and pharmaceuticals containing lipid bilayer particles containing such engineered polypeptides. 【0010】This disclosure is, in other words, one or more combinations of: [1] a first engineered polypeptide comprising a multimerizing sequence I and a cargo entity, and a second engineered polypeptide comprising a multimerizing sequence II that can associate with the multimerizing sequence I and an I-BAR (Inverse-Bin / Amphiphysin / Rvs) domain; [2] the combination according to [1] in which the multimerization of the multimerizing sequence II and the multimerizing sequence I is induced by a small molecule compound or light; [3] the combinations of the multimerizing sequence I and the multimerizing sequence II as follows: (1) GAI and GID1 (gibberellin insensitive dwarf 1); (2) abscisic acid insensitive 1 (ABI) and pyrabactin resistance-like (PYL) protein; (3) abscisic acid insensitive 1 (ABI) and PYL MandiThe combinations described in [1], which are one or more combinations selected from (4) FKBP and the FRB domain, (5) FKBP and Calcineurin A, (6) FKBP and CyP-Fas, (7) iLID and LOVssrA-SsrB, (8) LOVTRAP and LOV2-Zdk, and (9) the photoreceptor cryptochrome 2 (CRY2) and the CRY-interacting basic helix-loop-helix 1 (CIB1) protein. [4] The combination according to [1], wherein the I-BAR domain is one or more I-BAR domains selected from MIM, IRSp53, IRTKS, Pinkbar, and ABBA; [5] A polynucleotide encoding the first manipulated polypeptide according to [1], a polynucleotide encoding the second manipulated polypeptide according to [1], and one or more combinations of these polynucleotides; [6] One or more vectors comprising the combination according to [5]; [7] A host cell comprising the vector according to [6]; [8] A method for loading the first manipulated polypeptide according to [1] and the second manipulated polypeptide according to [1] into lipid bilayer particles, comprising culturing the host cell according to [7]; [9] The method according to [8], comprising culturing the host cell according to [7] in contact with a multimerization inducer;

[10] A method for producing lipid bilayer particles comprising the first manipulated polypeptide according to [1] and the second manipulated polypeptide according to [1], comprising recovering from the culture supernatant of the host cell according to [7];

[11] The following steps; The present invention provides a method for producing lipid bilayer particles comprising a first manipulated polypeptide and a second manipulated polypeptide according to [1], comprising the steps of (1) culturing host cells according to [7] in a culture medium in the presence of a polymerization-inducing factor, and (2) recovering the culture supernatant from the culture medium according to step (1); manipulated lipid bilayer particles comprising a first manipulated polypeptide and a second manipulated polypeptide according to [1], and a pharmaceutical product comprising the lipid bilayer particles according to

[13] . 【0011】In one non-limiting embodiment of this specification, an engineered polypeptide comprising a multimerizing sequence I and a cargo entity is provided. In some non-limiting embodiments, the multimerizing sequence I is directly or indirectly linked to the cargo entity. In some non-limiting embodiments, an engineered polypeptide is provided in which the multimerizing sequence I is directly linked to the cargo polypeptide via a peptide bond. In some non-limiting embodiments, preferred examples of cargo entities include synthetic nucleic acids, transcription factors, recombinases, base editors, prime editors, nucleases (e.g., TALEN, ZFN, etc.), kinases, kinase inhibitors, receptor signaling activators or inhibitors, in vivo, chromatin-modifying synthetic transcription factors, innate transcription factors, CRISPR-Cas family proteins, DNA molecules, RNA molecules, or ribonucleoprotein complexes. 【0012】 In another non-limiting embodiment of this specification, an engineered polypeptide is provided comprising an I-BAR (Inverse-Bin / Amphiphysin / Rvs) domain containing a multimerizing sequence II that can associate with a multimerizing sequence I. In some non-limiting embodiments, the multimerizing sequence II is directly or indirectly ligated to the I-BAR domain. In some non-limiting embodiments, an engineered polypeptide is provided in which the multimerizing sequence II is directly ligated to the I-BAR domain via a peptide bond. 【0013】In one non-limiting form, this specification provides an engineered polypeptide comprising (a) a multimerizing domain containing an FRB domain and (b) an I-BAR domain. In some embodiments, the engineered polypeptide may further comprise a linker that junctions the I-BAR domain and the multimerizing sequence containing the FRB domain. In some embodiments, the linker may include: (1) an amino acid sequence selected from SEQ ID NOs: 1 to 6, or (2) an amino acid sequence exhibiting at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6. The amino acid sequences of sequence numbers 1 through 6 are shown in Table 1 below. 【0014】In non-limiting embodiments of this specification, the disclosure provides engineered polypeptides comprising a multimerizing sequence I and a cargo entity, engineered polypeptides comprising a multimerizing sequence II and an I-BAR domain capable of multimerizing with multimerizing sequence I, and combinations of these engineered polypeptides. The disclosure also provides polynucleotides encoding engineered polypeptides comprising a multimerizing sequence I and a cargo entity, polynucleotides encoding engineered polypeptides comprising a multimerizing sequence II and an I-BAR domain capable of multimerizing with multimerizing sequence I, and one or more combinations of these polynucleotides, one or more vectors comprising one or more combinations of these polynucleotides, and host cells comprising one or more vectors. In some non-limiting embodiments, the host cell is a mammalian cell, which may be arbitrarily selected from HEK 293, HEK 293FS (Free Style), mesenchymal stem cells, meganuclear cells, induced pluripotent stem cells (iPSCs), T cells, erythrocytes, erythrocyte precursors, and iPSC-derived versions of any of the aforementioned cells. 【0015】In non-limiting embodiments herein, the disclosure provides a method for loading cargo entities into lipid bilayer particles (e.g., cell membrane-derived vesicles (CDMVs), particularly filopodium-derived vesicles (FDVs)), which includes expressing the engineered polypeptides disclosed herein in host cells. In some non-limiting embodiments, the culture of host cells expressing the engineered polypeptides includes contact with host cells of an inducer capable of inducing multimerization of the engineered polypeptide comprising multimerization sequence I and cargo entities, and the engineered polypeptide comprising multimerization sequence II and an I-BAR domain that can form a multimer with multimerization sequence I. Examples of non-limiting forms of contact with host cells of such inducers include irradiation of the host cells with blue light and contact with multimerization-inducing small molecule compounds. In some non-limiting embodiments, rapamycin and rapamycin analogs, gibberellins and gibberellin analogs, abscisic acid and abscisic acid analogs, and blue light irradiation are preferably exemplified as non-limiting forms of inducing factors. 【0016】 In some non-limiting embodiments herein, the disclosure provides a method for producing lipid bilayer particles, comprising recovering lipid bilayer particles containing cargo entities contained in the manipulated polypeptide (e.g., cell membrane-derived vesicles (CDMVs), particularly filopodium-derived vesicles (FDVs)) from the culture supernatant of a culture medium of host cells expressing the manipulated polypeptide disclosed herein. In some non-limiting embodiments, the culture method of the host cells expressing the manipulated polypeptide may be static culture, stirred culture, or shaking culture, as may be used. In some non-limiting embodiments, precipitation using crowding reagents, centrifugation, cross-flow filtration, column chromatography, affinity purification, etc., are preferably exemplified as methods for recovering and purifying the lipid bilayer particles. 【0017】 In non-limiting embodiments herein, the disclosure provides engineered polypeptides comprising a polymerizing sequence I and a cargo entity, engineered polypeptides comprising a polymerizing sequence II and an I-BAR domain capable of polymerizing with polymerizing sequence I, and lipid bilayer particles (e.g., cell membrane-derived vesicles (CDMVs), in particular filopodium-derived vesicles (FDVs)) comprising one or more combinations of these engineered polypeptides. 【0018】 In non-limiting embodiments herein, the disclosure provides pharmaceuticals containing engineered polypeptides comprising a polymerizing sequence I and a cargo entity, engineered polypeptides comprising a polymerizing sequence II and an I-BAR domain capable of polymerizing with polymerizing sequence I, and lipid bilayer particles (e.g., cell membrane-derived vesicles (CDMVs), particularly filopodium-derived vesicles (FDVs)) containing one or more combinations of these engineered polypeptides. In some non-limiting embodiments, suitable examples of pharmaceutical compositions included in the pharmaceuticals are sterile water, physiological saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and the like. 【0019】 The above general description and the following detailed description are illustrative and explanatory and are intended to provide further explanation of the claimed disclosure. Other claimed subjects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and the detailed description of the disclosure. 【0020】This figure shows cell migration induced by exogenous Rac1 in EVs. Figure 1(A) is a schematic diagram of the role of EVs in FBS in cell migration. FBS was inactivated by heating as in normal cell culture procedures, and EVs were removed by ultracentrifugation. Cell migration was measured using a wound healing assay. Figure 1(B) shows cell migration during the wound healing process of WT or CIP4 KO PANC-1 cells in the presence of FBS (top) or FBS with EVs removed (bottom). Cell ends at 0 hours (dotted line) and 12 hours (solid line) are shown. Scale bar is 100 μm. Figure 1(C) shows cell migration (4 × 10) in the presence of FBS, EV-deficient FBS, or EV-deficient FBS supplemented with l-EVs from FBS. 6 6 x 10 cells per 1 ml 8 Figure 1(D) shows the EVs (treated at EV / ml). The dynamin inhibitor Dynasore was applied at 40 μM. The cell migration region is the area of ​​injury occupied by migrating cells after 12 hours (4 replicates). Figure 1(D) shows the Rac1-containing EVs from HEK293 cell culture medium. Figure 1(E) shows cell migration (4 × 10) in the presence of l-EVs from MIM I-BAR-expressing HEK293 cells. 6 1.4 × 10 cells per 1 ml 9 Figure 1(F) shows Western blotting of Rac1 in the l-EV fraction and EV fraction from FBS of GFP or MIM I-BAR-expressing HEK293 cells. Number of EVs per lane is shown. Figure 1(G) shows Western blotting of MIM in the EV fraction from FBS. Figure 1(H) shows cell migration under l-EV from FBS treated with the Rac1 inhibitor EHT1864 at 20 μM (3 replicates). 4 × 10 6 6 x 10 cells per 1 ml 8Treated with EV / ml. Data are shown as mean ± SD. ***, p < 0.005; **, p < 0.01; *, p < 0.05. By one-way ANOVA with Tukey's HSD (honestly significant difference) test. Figure 2(A) shows the co-localization of l-EV containing Halo-Rac1 with the endosomal markers EEA1 and Lamp1. The dashed rectangle indicates magnification. Co-localization is indicated by arrows. Scale bar is 10 μm. Figure 2(B) shows the time course of co-localization by the Mander's coefficient in Figure 2(A) (45 cells with 3 replicates at each time point). Figure 2(C) shows the PALM imaging of l-EV expressing SF650B-Halo-Rac1 in late endosomes with Lamp1-mEos4b (left). The right is an enlarged view of the square region. The possibility of release of SF650B-Halo-Rac1 from endosomes is indicated by arrows. Scale bars are 10 μm (left) and 100 nm (right). Figure 2(D) shows the quantification of Halo-Rac1 release from endosomes in Figure 2(C). It shows the percentage of EVs in which Halo-Rac1 and Lamp1 cross over intracellularly (20 cells with 3 replicates, 5 EVs randomly selected per cell). Mean ± SD is shown. ***, p < 0.005. By two-sample equal variance two-sided Student's t-test. It is a figure showing the stoichiometry of Rac1 derived from EVs in recipient cells. Figure 3(A) shows Western blotting of WT or CIP4 KO PANC-1 cells (10 6 cells) treated with l-EV expressing MIM I-BAR and Halo-Rac1 (8 × 10 8 EV / ml) for the specified time, showing the result of quantifying internalized Halo-Rac1 against endogenous Rac1. l-EV expressing MIM I-BAR and Halo-Rac1 (5 × 10 8Blotting of EVs is also shown. Figure 3(B) shows the internalized Halo-Rac1 molecules per cell, calculated from Figures 3(A) and 12(B). Figure 3(C) shows the ratio of internalized Halo-Rac1 to endogenous Rac1, calculated from Figures 3(B) and 12(C). Figure 3(D) shows a summary of Rac1 molecule movement via EVs. Data are shown mean ± SD (3 replicates). *, p<0.05. One-way ANOVA with Tukey's HSD test. This figure shows the internalization of the designed EV cargo protein. Figure 4(A) is a schematic diagram of Cas12f incorporation into EVs and genome editing. Cas12f-Halo-FRB binds to FKBP-MIM in the presence of rapamycin and is loaded into MIM-dependent EVs. The reporter cassette contains mCherry and two types of frameshift GFP, expressing GFP after Cas12f-mediated cleavage of the target sequence and subsequent addition / deletion of nucleotides during repair. Figure 4(B) shows the results of Western blotting of HEK293 cells expressing mCherry-FKBP-MIM I-BAR and Cas12f-Halo-FRB and their l-EVs, with and without rapamycin treatment. The same number of cells (6 × 10⁶) 3 Cells) and EV (7×10 9 The lysates of the EV were analyzed. Figure 4(C) shows genome editing of reporter HEK293 cells by Cas12f-equipped EV. Cas12f reporter cells (HEK293 cells, 2.5 × 10⁶) 4 Cells) are divided into l-EV (1.5 × 10 in 0.2 ml) 10 Cells were treated with EV / ml. After 3 days, fluorescence was observed (left), and GFP-positive cells were quantified (right). EVs in cells expressing Cas12f and MIM 5KA were also examined. Figure 4(D) shows genome editing of PANC-1 reporter cells by Cas12f-carrying EVs. Cas12f reporter cells (PANC-1, 2.5 × 10⁶) 4 Cells) are divided into l-EV (1.5 × 10 in 0.2 ml) 10The cells were treated with EV (1.5 × 10⁶ per 0.2 ml). After 3 days, fluorescence was observed (left), and GFP-positive cells were quantified (right). EVs of cells expressing Cas12f and MIM 5KA were also examined. Figures 4(E) and 4(F) show the effects of heating or freezing on EVs. EV (1.5 × 10⁶ per 0.2 ml) 10 The cells were processed as shown (EV / ml), incubated with reporter HEK293 cells (E) and PANC-1 cells (F), and genome editing was performed as in Figures 4(C) and 4(D). Data are shown mean ± SD (6 replicates for Figure 4E, 3 replicates for the others). ***, p<0.005; *, p<0.05. One-way ANOVA with Tukey's HSD test. This figure shows the characteristics of the PANC-1 cell line. Figure 5(A) shows Western blotting of CIP4, GAPDH, Dynamin2, N-WASP, Cdc42, WAVE2, CLTA, and endophyllin A2 in PANC-1 cells, PANC-1 CIP4 KO cells, and CIP4 KO cells stably expressing CIP4-mEos4b or CIP4-mCherry. Figure 5(B) shows the localization of CIP4 in PANC-1 cells and the above cell lines during the wound healing process, by immunostaining of CIP4 or fluorescence of mCherry and mEos4b. Scale bar is 10 μm. This figure shows the characteristics of Rac1-containing EVs prepared from FBS. Figure 6(A) shows WT or CIP4 KO PANC-1 cells at the start of wound healing (0 hours) in the presence of FBS or FBS with EVs removed. Related to Figure 1B. Scale bar is 100 μm. Figure 6(B) shows the size distribution of EV particles in the l-EV and s-EV fractions prepared from FBS by NTA. Particle count is 10 4 Normalized by . Mean ± SD shown (from 5 measurements of the same sample). Figure 6(C) shows the total number of EVs in the l-EV fraction or s-EV fraction prepared from 1 ml of FBS. Mean ± SD shown (3 replicates). Figure 6(D) shows cell migration of PANC-1 cells in the presence of EV-removed FBS and s-EV fraction prepared from FBS. 12 hours, 4 × 10⁻⁶ 6 6 x 10 cells per 1 ml 8The cells were treated with EV / ml. Mean ± SD is shown (3 replicates). Figure 6(E) shows Rac1 contained within EVs. Effects of trypsination on Rac1 in l-EVs from MIM I-BAR-expressing HEK293 cells, purified Rac1 protein, and l-EVs from FBS. EVs or isoprotein amounts of protein were trypsinized, and the amount of Rac1 before and after trypsinization was quantified by Western blotting (left). The ratio of the amount of Rac1 after trypsinization to the amount of Rac1 before trypsinization is shown (right). Statistical significance was ***, p<0.005, by one-way ANOVA corrected for Tukey's HSD test. This figure shows Rac1 and its mutants in l-EV formation. Figure 7(A) shows the localization of Halo-Rac1 in WT, G12V, and T17N cells co-expressed with mCherry-MIM I-BAR in HEK293 cells. Scale bar is 10 μm. Figure 7(B) shows the size distribution of l-EVs co-expressing WT, G12V, or T17N Halo-Rac1 with mCherry-MIM I-BAR, as measured by NTA. The number of particles is 10. 4 Normalized (from 5 measurements of the same sample). Figure 7(C) shows the total number of l-EVs by NTA from HEK293 cells co-expressing WT, G12V, or T17N mutant Halo-Rac1 with mCherry-MIM I-BAR. Shows the number of EVs per cell (5 replicates). Figure 7(D) shows the number of l-EVs (1.6 × 10⁶) from cells co-expressing mCherry-MIM I-BAR and Halo-Rac1 mutants. 9 Figure 7(E) shows Western blotting of EVs (1.4 × 10¹⁶ cells) with Rac1 antibody. The arrows indicate the locations of Halo-Rac1 and endogenous Rac1, respectively. Quantification of Halo-Rac1 and Rac1 molecules per EV is shown on the right. Figure 7(E) shows cell migration in WT, G12V, or T17N mutants in the presence of l-EVs co-expressing Halo-Rac1 and mCherry-MIM I-BAR (5 replicates). Figure 7(F) shows MIM-expressing EVs (1.4 × 10¹⁶ cells). 9This shows cell migration in the presence of 5 μg of purified Rac1 protein, the same protein amount as EV (3 replicates). Data are shown as mean ± SD. Statistical significance was ***, p<0.005; **, p<0.01; *, p<0.05, calculated by one-way ANOVA with Tukey's HSD test. This figure shows EV uptake into recipient cells by CIP4. Left: Live imaging (4×10) of CIP4 KO PANC-1 cells expressing CIP4-mCherry (magenta) incubated with l-EV expressing GFP-MIM I-BAR (green). 6 Cells in 0.2 ml: 1.8 × 10 10 (Processed with EV / ml). Cell ends are indicated by dotted lines. Scale bar is 10 μm. Center: Time-lapse image of the region indicated by white dashed squares at 2.25-second intervals. Scale bar is 2 μm. Right: Line scan on the white dotted line in the middle image showing transient co-localization of MIM and CIP4-mCherry in EV. This figure shows intracellular transport of Rac1 in EV after uptake. Figure 9(A) is a schematic diagram of EV uptake. Figure 9(B) shows the internalization of l-EVs co-expressing mCherry-MIM I-BAR and SF650T-Halo-Rac1 into WT or CIP4 KO PANC-1 cells after 10 hours of culture. Puncta co-localized with mCherry-MIM I-BAR and SF650T-Halo-Rac1 are indicated by arrows. Scale bar is 5 μm. Figures 9(C) and 9(D) show 1.6 × 10⁶ cells per 1 ml. 9 10 EV / ml 6 Figures 9(E) and 9(F) show the time course of the percentage (9C) and number (9D) of internalized l-EVs after processing individual cells. Data are mean ± SD. Thirty cells were analyzed from three independent experiments. Figures 9(E) and 9(F) show 1.6 × 10⁶ cells per 1 ml. 9 10 EV / ml 6This figure shows the colocalization of Halo-Rac1 and l-EV expressing mCherry-MIM I-BAR with EEA1 (Figure 9E) or Lamp1 (Figure 9F) in WT cells after cell treatment and incubation for 3 hours. The arrows in the enlarged image indicate their colocalization. The scale bar is 10 μm. Figures 9(G) and 9(H) show the quantification of colocalization of EEA1 (Figure 9G) or Lamp1 (Figure 9H) and Halo-Rac1 in WT and CIP4 KO cells using Mander's coefficient after EV addition. The black bar line shows the mean value of 54 cells obtained from three independent experiments. Figure 9(I) shows the quantification of the relative position of endosomes and internalized EVs. Left: Diagram illustrating relative position measurement by the ratio of the distance between endosomes or EVs and the nuclear center (b) to the distance between the cell edge and the nuclear center (a). Right: Relative position of endosomes and EVs. The black bar represents the median value for each measurement, and the thin and thick dotted lines represent the median values ​​of early and late endosomes 3 hours after addition for 20 EVs or endosomes obtained from three independent experiments, respectively. Figure 9(J) shows the time course of cell migration in the presence of l-EVs expressing MIM I-BAR. 4×10 6 1.4 × 10 cells per 1 ml 9 Treatment was performed with EV / ml. Mean ± SD values ​​were obtained from six independent experiments. ***p < 0.005; **p < 0.01; *p < 0.05. Two-sample equal-variance two-sided Student's t-test. Figure shows the localization of exogenous Rac1 and lamellipodia formation. The left side of Figure 10(A) shows PANC-1 cells incubated with mCherry-MIM and l-EV with Halo or Halo-Rac1. 10 6 1.6 × 10⁶ cells per 1 ml 9Cells were treated with EV / ml for 12 hours. Arrows indicate the localization of Halo-Rac1 at the cell ends. Scale bar is 10 μm. The right side of Figure 10(A) shows the quantification of cells with Halo-Rac1 localized at the cell ends after incubation with EV for 3 hours. Data are shown as mean ± SD from three independent experiments. Statistical significance is ***, p < 0.005, by two-sample equal-variance two-sided Student's t-test. Figure 10(B) shows lamellipodia formation by l-EV induced by MIM expression. WT or CIP4 KO PANC-1 cells were incubated with l-EV expressing Halo-MIM I-BAR or Halo-only for 10 hours. 6 1.4 × 10⁴ cells per 1 ml 7 Cells were treated with EV / ml for 3 hours. After fixation, cells were stained with WAVE2 and lamellipodia were visualized (arrowheads). Scale bar is 40 μm. This figure shows exogenous Rac1 release from EVs and cell migration. Figure 11(A) shows the Halo-Rac1 signal per EV in endosomes by super-resolution observation. Halo-Rac1 signals forming clusters considered to be EVs within endosomes were counted (Figure 2C). The number of signals in 100 clusters from 3 replicates is shown. Figure 11(B) shows the localization of Halo-Rac1 in EV recipient cells during wound healing. PANC-1 cells treated with DMSO or bafilomycin were incubated with MIM I-BAR and l-EV expressing Halo-Rac1 for 3 hours, during which time 10 6 1.6 × 10⁶ cells per 1 ml 9 Cells were treated with EV / ml. Halo-Rac1 was visualized with Halo ligand. Cell outlines and wounds are shown by white and yellow dotted lines, respectively. Scale bar is 10 μm. Figure 11(C) shows the amount of Halo-Rac1 in the cytoplasm as detected by fluorescence. WT or CIP4 KO PANC-1 cells were incubated with l-EV expressing mCherry-MIM I-BAR and Halo-Rac1, and 10 6 1.6 × 10⁶ cells per 1 ml 9Cells were treated with EV / ml. The fluorescence intensity of Halo-Rac1 in the cytoplasm, excluding EV punctures, was quantified for 30 cells obtained from three independent experiments, compared to the mean value at 0.5 hours. The black bars indicate the mean values. Figures 11(D) to 11(F) show the effects of bafilomycin treatment. PANC-1 cells were treated with DMSO or bafilomycin and incubated with l-EVs expressing MIM I-BAR and Halo-Rac1. The quantification of cytoplasmic Halo-Rac1 intensity (Figure 11D), internalized Halo-Rac1-positive EVs (Figure 11E), and cell migration (Figure 11F) at 3 hours (Figure 11D, Figure 11E) and 12 hours (Figure 11F) is described. Data are mean ± SD from three independent experiments. Statistical significance was ***, p<0.005; **, p<0.01; *, p<0.05, calculated by two-sample equal-variance two-tailed Student's t-test (Figures 11A, 11C) or one-way ANOVA with Tukey's HSD test (Figures 11D-11F). This figure shows the quantification of Rac1 molecules from internalized EVs. Figure 12(A) shows cell migration by l-EVs derived from FBS (left) and 10% FBS (4×10). 7 (including individual l-EVs) or MIM l-EV (1.4 × 10⁻¹⁰) 9 A comparison of cell migration (right) by WT or CIP4 KO PANC-1 cells (4 × 10⁶). 6 Cells were treated with EV in 1 ml of culture medium. Figure 12(B) shows purified Rac1 (0.025 μg) and Halo-Rac1 / MIM-expressing l-EV (5 × 10⁻¹⁶). 7Figure 12(C) shows the amount of Halo-Rac1 and endogenous Rac1 in extracellular viable cells (EVs) measured by Western blotting. Figure 12(C) shows the amount of endogenous Rac1 molecules per cell measured by Western blotting as shown in Figure 3(A). Figure 12(D) is a diagram of the Halo-Rac1 sandwich ELISA. Figure 12(E) shows the calibration curve of absorbance at 450 nm in the ELISA based on Halo-Rac1 concentration. Figure 12(F) shows the quantification of endogenous Halo-Rac1 molecules per cell measured by ELISA. The results of measuring PANC-1 cells incubated with l-EVs expressing MIM I-BAR and Halo-Rac1 for 1 hour and 3 hours are shown. At that time, 6 × 10⁶ cells were measured in 6 ml of EV-removed FBS-containing medium. 6 Cells 1.5 × 10 9The cells were treated with EV / ml. Mean ± SD values ​​are shown (4 replicates). Figure 12(G) shows the amount of Rac1 molecules and the number of EVs in the EV source (top) and recipient cells (bottom). Mean ± SD values ​​are shown. "EV number per 1 ml FBS or medium" was measured by NTA. "Average Rac1 or Halo-Rac1 molecules per EV" was calculated from Figures 6(E) and 7(D) by comparing Rac1 in EV with purified Rac1. "Added EV number per recipient cell" was calculated by dividing the number of added EVs by the number of recipient cells. "Internalized Halo-Rac1 molecules per cell" is the Halo-Rac1 value of PANC-1 WT cells 3 hours after EV addition in Figure 3(B). "Internalized EV number per recipient cell" was calculated by dividing "Internalized Halo-Rac1 molecules per cell" by "Halo-Rac1 molecules per EV". "Percentage of internalized EVs to added EVs" was estimated as "Internalized EV number per recipient cell" / "Added EV number per recipient cell". This figure shows the quantification of internalized Rac1 molecules considering the ratio of Halo-Rac1-expressing EVs. Figure 13(A) shows the microscopic observation results of GFP-MIM I-BAR and Halo-Rac1 in l-EVs. l-EVs were labeled with SF650T Halo ligand to visualize the Halo and with PKH26 dye to visualize the lipid membrane. The scale bar is 10 μm. Figure 13(B) shows the quantification of Figure 13(A). It shows PKH26-positive EVs for fluorescence of GFP-MIM I-BAR and Halo-Rac1. EVs expressing GFP-MIM I-BAR alone or Halo-Rac1 alone (top) were measured, and a regression line was created for GFP-MIM / Halo-Rac1 co-expressing EVs (bottom). The regression line and the line shifted by +2.5 SD are shown as dotted and solid lines, respectively.The population with a deviation of 2.5 SD or greater was thought to possess either GFP-MIM I-BAR or Halo-Rac1. This was obtained from 2–3 × 10⁻⁶ samples from three independent experiments. 4The mean ± SD for each EV is shown. Figure 13(C) shows the fluorescence measurement of SF650T Halo ligand in droplets generated in oil. The measurement was performed under the same microscopic conditions as in Figure 13(A). The correlation between SF650T concentration and fluorescence intensity is shown below. Figure 13(D) shows the labeling efficiency of SF650T Halo ligand. Halo-Rac1 expressing cells were treated with SF650T at the same protein-to-ligand ratio as for EV labeling. The fluorescence intensity of the cell lysates was measured and compared with the fluorescence intensity of the SF650T Halo ligand solution. The Halo-Rac1 concentration in the cell lysates was determined by Western blotting using purified Rac1 as a control. Figure 13(E) shows the number of Halo-Rac1 molecules per MIM / Rac1 double-positive EV relative to EV size. Left: Relationship between Halo-Rac1 fluorescence intensity and concentration, according to Figure 13(C), taking into account that the ratio of fluorescence intensities of “SF650T ligand alone” and “SF650T ligand bound to HaloTag” is 0.076. Right: Plot of size, which is the diameter of the EV, against the fluorescence intensity of Halo-Rac1, according to Figure 13(B). Figure 13(F) shows the relationship between EV size per EV expressing GFP-MIM-I-BAR and Halo-Rac1 and the Halo-Rac1 molecule, calculated from Figure 13(E) and corrected for the labeling efficiency of the Halo ligand (24.4%, Figure 13D). Black bar: median value of each EV size. Halo-Rac1 values ​​for EVs with a size of less than 272 nm were summed, as described in “Materials and Methods” of the Examples. Figure 13(G) shows the estimated amount of Halo-Rac1 in MIM-I-BAR and Halo-Rac1-positive EVs, obtained by Western blotting and microscopic observation. This figure shows the quantification of Cas12f. Figure 14(A) shows the total number of l-EVs prepared from HEK293 cells co-expressing mCherry-FKBP-MIM I-BAR and Cas12f-Halo-FRB with or without 24-hour 10 nM rapamycin treatment. Mean ± SD shown (3 replicates). Figure 14(B) shows Western blotting of EVs expressing Halo-Rac1 or Cas12f-Halo-FRB for calculating the amount of Cas12f-Halo-FRB per EV.The number of loads for l-EVs expressing Halo-Rac1 and MIM I-BAR, and l-EVs expressing Cas12f-Halo-FRB and FKBP-MIM I-BAR, is 5 × 10, respectively. 7 and 5×10 9 It was EV. Figure 14(C) shows the stoichiometry of Cas12f in EVs internalized in reporter PANC-1 cells. (Table above) "Cas12f molecules per EV" was calculated from Figure 14(B) by comparing Cas12f-Halo in EVs with Halo-Rac1 and purified Rac1. "Added EV number per recipient cell" was calculated by dividing the number of added EVs by the number of recipient cells. "Percent of GFP-positive reporter PANC-1 cells" is the number of GFP-positive PANC-1 WT cells treated with Cas12f / MIM EVs divided by the number of observed cells (Figure 4D). "Internalized EV number" was calculated by multiplying "3.1±2.1% of added EVs," which is estimated to be the same as Rac1-containing EVs (Figure 3D), by "Added EV number per recipient cell." Estimated number of EVs received by genome-edited cells. (Below) The percentage of GFP-positive cells represents the EV number. The Gaussian distribution of “Internalized EV number per cell” has a mean of 3.7 × 10⁻⁶. 3 , with a standard deviation of 2.1 × 10 3This is shown by the blue line, and the population of GFP-positive cells (0.49%, shown in green) corresponds to 2.5 SD. "Internalized EV number per GFP-positive cell" was calculated as the value of "Internalized EV number per cell" at 2.5 SD of the Gaussian distribution. "Internalized Cas12f molecules per cell" was calculated by multiplying "Cas12f molecules per EV" by "Internalized EV number per GFP-positive cell". This figure shows the EV-derived Cas12f activity of several recipient cancer cells. Figure 15(A) shows GFP expression (left) and its quantification (right) in Cas12f reporter cells (HeLa cells) via genome editing with Cas12f-containing EVs after incubation for 3 days. Figure 15(B) shows GFP expression (left) and its quantification (right) in Cas12f reporter cells (Ca9-22 cells) via genome editing with Cas12f-containing EVs after incubation for 3 days. Figure 15(C) shows GFP expression in Cas12f reporter cells (CIP4 KO PANC-1 cells) via genome editing with Cas12f-containing EV after incubation for 3 days. HeLa cells (A), Ca9-22 cells (B), and CIP4 KO PANC-1 cells (C) were used as Cas12f reporter cells, with a sample size of 0.3 × 10⁶. 10 EV 2.5 × 10 in 0.2 ml of culture medium 4The vectors were added to recipient cells. Scale bar is 100 μm. Mean ± SD is shown (3 replicates). ***, p<0.005. One-way ANOVA with Tukey's HSD test. This figure shows that EVs derived from protrusions are superior to EVs derived from endosomes in Cas12f delivery. Figure 16(A) shows Western blotting of l-EVs and s-EVs from HEK293 cells co-transfected with mCherry-FKBP-MIM I-BAR- or CD63-GFP-FKBP expression vectors and Cas12f-Halo-FRB co-expressing cells, with or without rapamycin treatment. Cells transfected with a vector expressing Cas12f followed by the T2A sequence and I-BAR domain were also analyzed. The same number of cells (6 × 10) 3 Cells) and EV (7×10 9 The lysates of EV were analyzed. Figure 16(B) shows the quantification in Figure 16(A). Figure 16(C) shows genome editing of reporter cells by Cas12f-supported EV. Cas12f reporter HEK293 cells (2.5 × 10⁻¹⁰) 4 Cells) are divided into l-EV (1.5 × 10 in 0.2 ml) 10Cells were treated with EV / ml. After 3 days, fluorescence was observed and the number of GFP-expressing cells was examined. Figure 16(D) shows the genome editing efficiency of EVs. Efficiency was calculated by dividing the number of GFP-positive cells by the amount of Cas12f per EV. Data are shown as mean ± SD (3 replicates). ***, p<0.005; *, p<0.05, and a one-way ANOVA with Tukey's HSD test was performed. Figure 17(A) shows the number of EVs (relative value) in the Large EV fraction obtained by culturing B16 cells, Lenti-X (HEK293 derivative) cells, and HEK293A (HEK293 adherent) cells under different culture conditions. Figure 17(B) shows the number of EVs (relative value) in the Large EV fraction obtained by culturing FS293 (FreeStyle 293) cells under different culture conditions. Figure 18(A) shows the relative number of EVs in the Small EV fraction obtained by culturing B16 cells, Lenti-X cells, and HEK293A cells under different culture conditions. Figure 18(B) shows the relative number of EVs in the Small EV fraction obtained by culturing FS293 cells under different culture conditions. Figure 19(A) shows the number of EVs per cell in the Large EV fraction obtained by culturing B16 cells, Lenti-X cells, and HEK293A cells under different culture conditions. Figure 19(B) shows the number of EVs per cell in the Large EV fraction obtained by culturing Lenti-X cells and HEK293A cells under different culture conditions. Figure 19(C) shows the number of EVs per cell in the Large EV fraction obtained by culturing FS293 cells under different culture conditions. Figure 20(A) shows the number of EVs per cell in the Small EV fraction obtained by culturing B16 cells, Lenti-X cells, and HEK293A cells under different culture conditions. Figure 20(B) shows the number of EVs per cell in the Small EV fraction obtained by culturing FS293 cells under different culture conditions. 【0021】 To provide a substantial understanding of this technology, please understand that several forms, modes, examples, variations, and features of this disclosure are described in detail below. 【0022】In implementing this disclosure, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology, and recombinant DNA will be used. For example, Sambrook and Russell eds (2001)Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al, eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach, Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.See (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology. 【0023】 (Definitions) Unless otherwise specified or indicated by context, the terms “a,” “an,” and “the” mean one or more. For example, a fusion protein, an extracellular vesicle, and a cell should be interpreted as meaning one or more fusion proteins, one or more extracellular vesicles, one or more extracellular vesicles, and one or more cells, respectively. 【0024】 Where used herein, about, approximately, substantially, and significantly may be understood by those skilled in the art and may vary to some extent depending on the context in which they are used. In cases where there is use of these terms that is not obvious to those skilled in the art, given the context in which they are used, about and approximately mean plus or minus about 1% of the given term, substantially and significantly mean plus < 10% or minus > 10% of the given term. 【0025】Where used herein, a control is an alternative sample used in an experiment for comparative purposes. A control may be a positive control or a negative control. The purpose of such an experiment is, for example, to determine the correlation between the effectiveness of loading cargo entities, such as cargo proteins or cargo nucleic acids, into lipid bilayer particles and the composition or structure of those cargo entities. A positive control (a cargo entity, such as a cargo protein or cargo nucleic acid, known to exhibit the desired loading effectiveness) and a negative control (a cargo entity, such as a cargo protein or cargo nucleic acid, that is not loaded into lipid bilayer particles) are typically used. 【0026】 As used herein, the term extracellular vesicle (EV) is interpreted to include all nanometer-scale lipid bilayer particles that are "secreted" and "budding" by cells, such as "exosomes" and "cell membrane-derived vesicles." As used herein, the term "exosome" refers to extracellular vesicles that originate from internal endocytic compartments and multi-vesicular bodies. The term "cell membrane-derived vesicles (CDMV)" refers to vesicles that are secreted or budding from the cell membrane. In particular, among cell membrane-derived vesicles, "filopodium-derived vesicles (FDV)" refer to vesicles that budding directly from the cell surface. Extracellular vesicles, as well as their isolation and analysis, can be carried out, for example, by referring to Doyle et al., (Cells. 2019 Jul 15;8 (7), 727 (2019)), which is well known to those skilled in the art. 【0027】As used herein, the term "engineered" refers to an embodiment designed, produced, and / or manipulated by human hands. Herein, a polynucleotide is defined, for example, as engineered when (i) two or more polynucleotide sequences that are not naturally linked to each other are designed or otherwise caused by human hands to be directly linked; (ii) and / or a particular residue in the polynucleotide becomes a residue that does not exist in nature; or (iii) it is linked by human hands to an entity or region that is not naturally linked. For example, in some non-limiting embodiments described and / or utilized herein, an engineered polynucleotide is linked by human hands such that a regulatory sequence that is operationally related to a first coding sequence but not to a second coding sequence is operationally related to the second coding sequence. Conversely, in some embodiments herein, a polypeptide is defined as an engineered polypeptide if it is encoded by or expressed from an engineered polynucleotide, and / or if a peptide is produced in a manner other than in its natural expression within a cell. Similarly, a cell or organ is defined herein as an engineered cell or organ if a host cell is engineered in such a way that its genetic, epigenetic, and / or phenotypic identity is altered compared to a suitable reference cell, such as an unengineered identical host cell.In some embodiments herein, the operation is or includes genetic manipulation, resulting in alteration of its genetic information (for example, when new genetic material that did not previously exist is introduced, for example, by transformation, conjugation, somatic hybridization, transfection, transduction, or other mechanisms, or when pre-existing genetic material is modified or removed, for example, by substitution or deletion mutation, or by a conjugation procedure). Even if the actual operation is performed on ancestral polynucleotides or cells, as is understood by those skilled in the art, the offspring of the manipulated polynucleotide or manipulated cell are still defined herein as the manipulated polynucleotide or manipulated cell. 【0028】 As used herein, “engineered lipid bilayer particles” refers to lipid bilayer particles that have been engineered as described herein. For example, in some embodiments, lipid bilayer particles are defined herein as engineered lipid bilayer particles when they are produced synthetically, i.e., not by cells. In other non-limiting forms herein, lipid bilayer particles are defined as engineered lipid bilayer particles when they have been produced by engineered cells. In some non-limiting embodiments, engineered lipid bilayer particles are produced by engineered cells to contain a first engineered polypeptide containing a multimerization sequence I and cargo entities, and a second engineered polypeptide containing a multimerization sequence II and an I-BAR domain that can associate with multimerization sequence I. As a result, lipid bilayer particles containing CDMV and FDV produced by such engineered cells contain significantly more cargo entities than equivalent lipid bilayer particles produced by reference cells. 【0029】 As used herein, the term “gene” means a DNA segment containing all the information about the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other non-translational regions that control expression. 【0030】As used herein, “homology,” “identity,” or “similarity” refers to the similarity of sequences between two peptides or between two nucleic acid entities. Homology can be determined by comparing positions within each sequence that can be aligned for comparison. Entities are homologous at a position if that position within the sequences being compared is occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The “sequence identity” of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with respect to another sequence (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. The percentage of alignment and homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters may also be used as appropriate for alignment purposes. One alignment program, in non-limiting embodiments, is BLAST, which uses default parameters. In non-limiting embodiments, BLASTN and BLASTP may be preferred, using the following parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sort = HIGH SCORE; database = co-occurrence expression non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissPro + SPupdate + PIR. 【0031】Details of these programs can be found at the National Center for Biotechnology Information (NCBI). Bioequivalent polynucleotides are those that have a specified percentage of homology and encode polypeptides with the same or similar biological activity. If two sequences have less than 40% identity, or less than 25%, they are considered "unrelated" or "non-homologous." 【0032】 As used herein, the terms “include” and “including,” as well as “contain” and “containing,” have the same meaning as “comprise” and “comprising,” which are interpreted as “open” transitional clauses and do not limit claims to the enumerated elements following these transitional clauses. The term “consisting of” is included in the term “include,” but should be interpreted as a “closed” transitional clause and limits claims to the enumerated elements following this transitional clause. The term “consisting essentially of” is included in the term “include,” but should be interpreted as a “partially closed” transitional clause and allows for additional elements following this transitional clause, provided that such additional elements do not substantially affect the basic and novel features of the claim. 【0033】 As used herein, the terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid,” and “nucleic acid sequence” refer to nucleotides, oligonucleotides, polynucleotides (these terms may be used interchangeably), or any fragment thereof. These terms also refer to DNA or RNA (which may be single-stranded or double-stranded, and which may be sense strands or antisense strands) of genomic, naturally occurring, or synthetic origin. 【0034】With respect to polynucleotide sequences, the terms “percent identity” and “% identity” refer to the percentage of matching residues between at least two polynucleotide sequences aligned using a standardized algorithm. Such algorithms optimize the alignment between two sequences by inserting gaps into the sequences being compared in a standardized and reproducible manner, thus achieving a more meaningful comparison of the two sequences. Percent identity of nucleic acid sequences can be determined as understood in the art (see, for example, U.S. Patent No. 7,396,664). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI website in Bethesda, Maryland. The BLAST software suite includes various sequence analysis programs, including “blastn,” which is used to align known polynucleotide sequences with other polynucleotide sequences from various databases. A tool called “BLAST 2 Sequence” is also available, which is used to directly compare two nucleotide sequences. The "BLAST 2 Sequencing" tool is interactively accessible and available on the NCBI website. The "BLAST 2 Sequencing" tool can be used with both blastn and blastp, as described above. 【0035】With respect to polynucleotide sequences, percentage identity may be measured over the entire length of a defined polynucleotide sequence, such as defined by a particular sequence number, or over a shorter length, such as the length of a fragment taken from a larger defined sequence, for example, over the length of a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 consecutive nucleotides. Such lengths are merely illustrative, and it is understood that the length of any fragment supported by sequences shown herein, in tables, figures, or sequence listings may be used to illustrate the length over which the percentage of identity can be measured. 【0036】 With respect to polynucleotide sequences, a “variant,” “mutant,” or “derivative” can be defined using BLAST with the “BLAST2 sequence” tool available on the National Center for Biotechnology Information website as a nucleic acid sequence that has at least 50% sequence identity with one of a particular nucleic acid sequences over a certain length (see TA Tatusova et al., FEMS Microbiol Lett. 174, 247-250 (1999)). Such pairs of nucleic acids may exhibit, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity over a specific defined length. 【0037】Even nucleic acid sequences that do not exhibit high levels of identity may code for similar amino acid sequences because, due to the degeneracy of the genetic code, a single amino acid can be coded by multiple codons. It is understood in this art that this degeneracy can be used to modify nucleic acid sequences and generate multiple nucleic acid sequences that code for substantially the same protein. For example, the polynucleotide sequences understood here may code for a protein, and the codons may be optimized for expression in a particular host. In this art, codon frequency tables have been created for numerous host organisms, including humans, mice, rats, pigs, E. coli, plants, and other host cells. 【0038】 In relation to polynucleotide sequences, a "recombinant nucleic acid" is a sequence that does not exist naturally, or a sequence created by artificially combining two or more isolated sequence segments. This artificial combination can be achieved by chemical synthesis, but more commonly, by artificially manipulating isolated segments of nucleic acids, for example, by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that have been modified by adding, substituting, or deleting parts of the nucleic acid. Recombinant nucleic acids often contain nucleic acid sequences that are functionally linked to a promoter sequence. Such recombinant nucleic acids may be part of a vector used, for example, to transform cells. 【0039】 The nucleic acids disclosed herein may be “substantially isolated or purified.” The term “substantially isolated or purified” means nucleic acids taken from the natural environment that are free from at least 60%, at least 75%, at least 90%, or at least 95% of other naturally occurring components. 【0040】"Transformed" or "transfected" refers to the process by which a foreign nucleic acid (e.g., DNA or RNA) is introduced into a recipient cell. Transformation or transfection may be carried out under natural or artificial conditions according to a variety of methods well known in the art, and may rely on any known method for inserting a foreign nucleic acid sequence into a host cell, such as a prokaryote or eukaryote. The method of transformation or transfection is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection or nonviral delivery. Methods of nonviral delivery of nucleic acids include lipofection, nucleofection, microinjection, electroporation, heat shock, particle bombardment, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid nucleic acid complexes, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Patent No. 6,623,323. Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described in WO1991 / 017424, WO1991 / 016024, etc. Delivery can be made to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). The terms “transformed cells” or “transfected cells” include stably transformed or transfected cells in which the inserted DNA can replicate as an autonomously replicating plasmid or as part of a host chromosome, as well as ephemerally transformed or transfected cells in which the inserted DNA or RNA expresses for a limited period. In another embodiment, the term also includes stably transfected cells. 【0041】The polynucleotide sequences discussed herein may be present in an expression vector. For example, a vector may contain (a) a polynucleotide encoding an ORF of multimerization sequence I, and (b) a polynucleotide encoding an I-BAR domain, for example, an I-BAR domain selected from the group consisting of MIM, IRSp53, IRTKS, Pinkbar, and ABBA. The vector may also contain (c) a polynucleotide encoding an ORF of multimerization sequence II that can associate with multimerization sequence I, and (b) a polynucleotide expressing any cargo entity. The polynucleotides present in the vector may be operably ligated to a prokaryotic or eukaryotic promoter. "Operatably ligated" means that a first nucleic acid sequence is in a functional relationship with a second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably ligated to the coding sequence. The operably ligated DNA sequences may be adjacent or contiguous, and may be in the same reading frame if it is necessary to ligate two protein coding regions. The vectors envisioned here may include heterologous promoters (e.g., eukaryotic or prokaryotic promoters) operably ligated to a protein-coding polynucleotide. A "heterologous promoter" refers to a promoter that is not the native or endogenous promoter of the protein or RNA being expressed. 【0042】 As used here, "expression" refers to the process by which polynucleotides are transcribed from a DNA template (such as mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and their encoded polypeptides are sometimes collectively referred to as "gene products." When polynucleotides originate from genomic DNA, expression may include mRNA splicing in eukaryotic cells. 【0043】The term "vector" refers to a means by which nucleic acids (e.g., DNA) can be introduced into a host organism or host tissue. There are various types of vectors, including plasmid vectors, bacteriophage vectors, cosmid vectors, bacterial vectors, and viral vectors. As used herein, "vector" refers to recombinant nucleic acids designed to express heterologous polypeptides (e.g., the manipulated polypeptides disclosed herein). Recombinant nucleic acids typically contain a cis-acting element for the expression of the heterologous polypeptide. 【0044】 Any conventional vector used for expression in eukaryotic cells can be used to directly introduce DNA into the target. Expression vectors containing eukaryotic viral regulatory elements can be used as eukaryotic expression vectors (e.g., vectors containing SV40, CMV, or retroviral promoters or enhancers). Non-exclusive exemplary vectors include vectors that express proteins under the direction of promoters such as the SV40 early promoter, SV40 late promoter, metallothionein promoter, human cytomegalovirus promoter, mouse mammary cancer virus promoter, and Rous sarcoma virus promoter. Expression vectors considered herein include eukaryotic or prokaryotic regulatory sequences that regulate the expression of heterologous proteins (e.g., engineered polypeptides disclosed herein). Prokaryotic expression regulatory sequences may include constitutive or inductive promoters (e.g., T3, T7, Lac, trp, phoA), ribosome binding sites, or transcription terminators. 【0045】The vectors contemplated herein can be introduced into and propagate in prokaryotes and used to amplify copies of the vector to be introduced into eukaryotic cells, or as intermediate vectors in the production of vectors to be introduced into eukaryotic cells (e.g., to amplify plasmids as part of a viral vector packaging system). Prokaryotes can be used to amplify copies of the vector and express one or more nucleic acids, for example, to provide a source of one or more proteins for delivery to a host cell or host organism. Protein expression in prokaryotes can be carried out using E. coli with a vector containing a constitutive or inducible promoter that induces the expression of either a protein or a fusion protein containing a protein or a functional fragment thereof. The fusion vector adds several amino acids to the protein it encodes, such as the amino terminus of a recombinant protein. Such fusion vectors are used to (i) increase the expression of recombinant proteins, (ii) enhance the solubility of recombinant proteins, (iii) aid in the purification of recombinant proteins by acting as ligands in affinity purification (e.g., His tags), (iv) tag recombinant proteins for identification (e.g., green fluorescent protein (GFP) or antigens recognizable by labeled antibodies (e.g., HA)), and (v) promote the localization of recombinant proteins to specific regions of cells [e.g., when the protein is fused to a nuclear localization sequence (NLS) (e.g., its N-terminus or C-terminus), which may include the NLS of SV40 (PKKKRRV (SEQ ID NO: 7)), nuclear plasmin (KRPAATKKAGQAKKKK (SEQ ID NO: 8)), C-myc, the M9 domain of hnRNP A1, or synthetic NLS]. The importance of neutral and acidic amino acids in NLS has also been studied (see Makkerh et al., Curr Biol 6, 8, 1025-1027 (1996)), and it can fulfill one or more of these objectives.In many cases, fusion expression vectors incorporate proteolytic cleavage sites at the junction of the fusion region and the recombinant protein, allowing for the separation of the recombinant protein from the fusion region after purification of the fusion protein. Examples of such enzymes and their homologous recognition sequences include factor Xa, thrombin, and enterokinase. 【0046】 Methods disclosed herein may include expressing in host cells one or more vectors described herein, one or more polynucleotides, one or more transcripts thereof, and / or one or more proteins transcribed therefrom. Furthermore, host cells produced by such methods, and organisms (such as animals, plants, or fungi) containing or produced from such cells are envisioned. Lipid bilayer particles containing extracellular vesicles disclosed herein can be prepared by introducing vectors expressing mRNA and cargo RNA encoding the manipulated polypeptides described herein. Nucleic acids can be introduced into mammalian cells or target tissues using conventional viral and nonviral-based gene transfer methods. Nonviral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids compounded with delivery media such as liposomes. Viral vector delivery systems include DNA viruses and RNA viruses that have either an episomal genome or an integrated genome after delivery to cells. 【0047】In the methods contemplated herein, host cells can be transiently transfected or non-transiently (i.e., stably) transduced with one or more vectors described herein. In some embodiments, host cells are transfected in a manner that occurs spontaneously within a subject (i.e., in vivo). In some embodiments, the cells to be transfected are taken from a subject (i.e., explanted). In some embodiments, the cells are derived from cells taken from a subject such as a cell line. Suitable cells may include stem cells (e.g., embryonic stem cells and pluripotent stem cells). Cells transfected with one or more vectors described herein can be used to establish new cell lines containing sequences derived from one or more vectors. In the methods contemplated herein, components such as vectors containing polynucleotides encoding the manipulated polypeptides described herein can be transiently transfected into cells (e.g., transient transfection of one or more vectors, or transfection of RNA), modified via the activity of the complex, and new cell lines containing cells with the modification but lacking other exogenous sequences can be established. 【0048】 As used herein, the terms “protein,” “polypeptide,” or “peptide” may be used interchangeably to refer to polymers of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, usually with a length of 50, 60, 70, 80, 90, or 100 amino acids or more. A “peptide” is defined as a shorter polymer of amino acids, usually with a length of 50, 40, 30, or 20 amino acids or less. 【0049】The “proteins” as envisioned herein typically comprise polymers of natural or non-natural amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). The proteins envisioned herein may be further modified in vitro or in vivo to include non-amino acid moieties. These modifications include acylation (e.g., O-acylation (ester), N-acylation (amide), S-acylation (thioester)), acetylation (e.g., addition of an acetyl group at either the N-terminus or a lysine residue of a protein), formylation, lipoylation (e.g., addition of lipoic acid, a C8 functional group), myristoylation (e.g., addition of myristic acid, a C14 saturated acid), palmitoylation (e.g., addition of palmitic acid, a C16 saturated acid), alkylation (e.g., addition of alkyl groups such as methyl at lysine or arginine residues), isoprenylation or prenylation (e.g., farnesol or geranylgone). This is different from glycation, polysialylation (e.g., addition of polycyanate), polysylation (e.g., addition of polycyanate), glycosylation (e.g., formation of a glycosyl group on asparagine, hydroxylysine, serine, or threonine), which are considered non-enzymatic additions of sugars, polysialylation (e.g., addition of polysialylate), glycopiation (e.g., formation of a glycosylphosphatidylinositol (GPI) anchor, hydroxylation, iodization (e.g., of thyroid hormones), and phosphorylation (e.g., addition of a phosphate group, usually on serine, tyrosine, threonine, or histidine), which are considered non-enzymatic additions of sugars. 【0050】With respect to proteins, the term "amino acid residue" may also include amino acid residues selected from the group consisting of homocysteine, 2-aminoadipic acid, N-ethylasparagine, 3-aminoadipic acid, hydroxylysine, β-alanine, β-aminopropionic acid, allo-hydroxylysine, 2-aminobutyric acid, 3-hydroxyproline, 4-aminobutyric acid, 4-hydroxyproline, piperidine, 6-aminocaproic acid, isodesmosine, 2-aminoheptanoic acid, allo-isoleucine, 2-aminoisobutyric acid, N-methylglycine, sarcosine, 3-aminoisobutyric acid, N-methylisoleucine, 2-aminopimelic acid, 6-N-methyllysine, 2,4-diaminobutyric acid, N-methylvaline, desmosine, norvaline, 2,2'-diaminopimelic acid, norleucine, 2,3-diaminopropionic acid, ornithine, and N-ethylglycine. 【0051】 The proteins disclosed herein include “wild-type” proteins and their variants, mutants, and derivatives. As used herein, the term “wild-type” is a technical term understood by those skilled in the art and refers to a typical form of an organism, strain, gene, or trait occurring in nature, distinct from mutant or variant forms. As used herein, “variant,” “mutant,” or “derivative” refers to a protein entity having a different amino acid sequence from a reference protein or polypeptide entity. A mutant or mutant may have one or more insertions, additions, deletions, or substitutions of amino acid residues compared to the reference entity. A mutant or mutant may contain a functional fragment of the reference entity. For example, a mutant or variant entity may have one or more insertions, additions, deletions, or substitutions of amino acid residues compared to the reference polypeptide. 【0052】In the context of proteins, the term "deletion" refers to the absence of one or more amino acid residues due to a change in the amino acid sequence. A deletion involves the removal of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acid residues, or a range of amino acid residues delimited by any of these values ​​(e.g., a deletion of 5 to 10 amino acids). Deletions include internal deletions or terminal deletions (e.g., N-terminal or C-terminal cleavage of a reference polypeptide). "Variants," "mutants," or "derivatives" of a reference polypeptide sequence may contain deletions relative to the reference polypeptide sequence. 【0053】In relation to proteins, the term "fragment" refers to a portion of an amino acid sequence that is identical in sequence to a reference sequence but shorter in length. A fragment can be as long as the full length of the reference sequence minus at least one amino acid residue. For example, a fragment can contain 5 to 1000 consecutive amino acid residues of the reference polypeptide. In some embodiments, a fragment can contain at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 consecutive amino acid residues of the reference polypeptide. In other embodiments, a fragment can contain fewer than approximately 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 consecutive amino acid residues of the reference polypeptide. Or, in other embodiments, a fragment has a length within a range limited by any of these values ​​(e.g., a range of 50 to 100 consecutive amino acids of the reference polypeptide). Fragments can be preferentially selected from specific regions of an entity. The term “at least a fragment” encompasses the full-length polypeptide. Fragments can include N-terminal cleavage, C-terminal cleavage, or both cleavage of the full-length protein. Among the “fragments,” those that have a different amino acid sequence and number of amino acids from the reference polypeptide but can have at least one of the same functions as the reference polypeptide are referred to herein as “functional fragments.” “Variants,” “mutants,” or “derivatives” of a reference polypeptide sequence can include fragments of the reference polypeptide sequence. 【0054】In relation to proteins, the terms "insertion" and "addition" refer to the addition of one or more amino acid residues through a change in the amino acid sequence. Insertions or additions refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acid residues, or a range of amino acid residues delimited by any of these values ​​(for example, an insertion or addition of 5 to 10 amino acids). A "variant," "mutant," or "derivative" of a reference polypeptide sequence includes insertions or additions to the reference polypeptide sequence. Protein variants include N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions, and internal insertions. 【0055】 With respect to proteins, as used herein, “chimeric protein,” “chimeric peptide,” “fusion protein,” or “fusion peptide” refers to a protein or peptide created by linking two or more functional domains from separate or identical proteins via an amino acid linker, resulting in a single polypeptide possessing functional properties derived from each of the original proteins. The linker is 10 to 50 amino acids long and is rich in glycine for flexibility and serine or threonine for solubility. A “variant” of a reference polypeptide sequence may include a fusion polypeptide containing the reference polypeptide. 【0056】In relation to proteins, the terms "percent identity" and "% identity" refer to the percentage of residue agreement between at least two amino acid sequences aligned using a standardized algorithm. Methods for amino acid sequence alignment are well known. Some alignment methods consider conserved amino acid substitutions. Such conserved substitutions, described in more detail below, typically maintain the charge and hydrophobicity of the substitution site, and therefore the structure (and thus function) of the polypeptide. The percentage identity of amino acid sequences can be determined as understood in the art (see, for example, U.S. Patent No. 7,396,664). The National Center for Biotechnology (NCBI) Basic Local Alignment Search Tool (BLAST) provides a set of commonly used and freely available sequence comparison algorithms. This tool is available from several sources, including the NCBI website (Bethesda, Maryland). The BLAST software suite includes various sequence analysis programs, such as "blastp," used to align known amino acid sequences with other amino acid sequences from various databases. As described herein, a variant, mutant, or fragment (e.g., a protein variant, mutant, or fragment thereof) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% amino acid sequence identity compared to a reference entity. 【0057】With respect to proteins, the identity percentage may be measured over the length of the entire defined polypeptide sequence, for example, as defined by a specific sequence number, or over a shorter length, for example, the length of a fragment obtained from a larger defined polypeptide sequence, for example, a fragment of at least 1, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues. Such lengths are merely illustrative, and it is understood that the length of any fragment supported by sequences shown herein, in tables, figures, or sequence listings can be used to describe the length over which the identity percentage can be measured. 【0058】 With respect to proteins, the amino acid sequences of the variants, mutants, or derivatives considered herein may contain conserved amino acid substitutions to the reference amino acid sequence. For example, variant, mutant, or derivative proteins may contain conserved amino acid substitutions to the reference entity. A “conserved amino acid substitution” is a substitution to another amino acid that is predicted to interfere least least with the properties of the reference polypeptide. In other words, a conserved amino acid substitution substantially preserves the structure and function of the reference polypeptide. The following table lists exemplary conserved amino acid substitutions considered herein. 【0059】 Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the substituted region (e.g., a beta-sheet or alpha-helix structure), (b) the charge or hydrophobicity of the entities at the substituted site, and / or (c) the bulkiness of the side chains. 【0060】Proteins, variants, mutants, and derivatives disclosed or described herein may have one or more functional or biological activities demonstrated by a reference polypeptide (e.g., one or more functional or biological activities demonstrated by a wild-type protein). For example, a protein, variant, mutant, or derivative containing a disclosed I-BAR domain may have one or more biological activities, including localization to lipid bilayer particles (e.g., cell membrane-derived vesicles (CDMVs), particularly filopodium-derived vesicles (FDVs)). 【0061】 The disclosed proteins can be substantially isolated or purified. The term “substantially isolated or purified” means a protein that has been removed from its natural environment and is free from at least 60%, at least 75%, at least 90%, or at least 95% of other components that are naturally related to each other. 【0062】 For example, with respect to cells, nucleic acids, proteins, or vectors, the term “recombinant” as used herein means that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by modification of a native nucleic acid or protein, or that the material for which it is derived is a cell that has been thus modified. Thus, for example, a recombinant cell will express genes that are not present in the cell’s native (non-recombinant) form, or will abnormally express native genes that would otherwise be expressed or not expressed at all. 【0063】As used herein, the terms “target domain” or “target peptide” refer to a peptide portion that facilitates the specific binding of lipid bilayer particles (e.g., cell membrane-derived vesicles (CDMVs), and especially filopodium-derived vesicles (FDVs)) to target cells. Sample “target domain” or “target peptide” includes, but is not limited to, antibodies and any antibody or antigen-binding fragments, such as Fab, Fab' and F(ab')2, Fd, single-chain Fvs(scFv), single-chain antibodies, disulfide-bonded Fvs(sdFv), novelly designed binding molecules, affinibodies, DARPIN, and nanobodies. These antibody fragments are well known to those skilled in the art. 【0064】 (Detailed Description of Embodiments) In one non-limiting form of this specification, the disclosure provides a first engineered polypeptide comprising (a) a polymerizing sequence I and a cargo entity. In another non-limiting form of this specification, the disclosure provides a second engineered polypeptide comprising (b) a polymerizing sequence II and an I-BAR domain that can form a polymer with polymerizing sequence I. 【0065】 The cargo entity and the I-BAR domain are linked by multimerization through the binding of multimerization sequence I in the first manipulated polypeptide to multimerization sequence II in the second manipulated polypeptide. Although not intended to be constrained by any particular theory, this allows for the localization of the multimer containing the cargo entity to the cell membrane, particularly to the cell protrusions. This localization of the cargo entity makes it possible to increase the amount of cargo entity within the lipid bilayer particles formed from the cell membrane and cell protrusions. The lipid bilayer particles thus formed contain a high concentration of the desired cargo entity and have low contamination from impurities and denatured proteins. Furthermore, the lipid bilayer particles have low immunogenicity derived from foreign proteins of viral origin. Therefore, the desired cargo entity can be efficiently delivered to cells by lipid bilayer particles with low immunogenicity. 【0066】 While not intended to be constrained by any particular theory, if cargo entities are localized to cellular protrusions, the cellular protrusions can be cleaved by shaking culture, and lipid bilayer particles can be prepared from the culture medium by short-duration centrifugation at normal speed (without using ultracentrifugation). 【0067】(Multimerization Sequences I and II) There are no particular restrictions on the combination of multimerization sequence I and multimerization sequence II. A person skilled in the art can appropriately select one or more combinations that can achieve the desired objective, taking into consideration the properties of the factors that induce multimerization. As an unspecified form of the factors that induce multimerization in this specification, light or low molecular weight compounds are preferably exemplified. Some non-limiting forms of such combinations of multimerizing sequence I and multimerizing sequence II, and the factors that induce multimerization, include: (1) a combination of the photoreceptor cryptochrome 2 (CRY2), which can be multimerized by blue light, and the CRY-interacting basic helix-loop-helix 1 (CIB1) protein, which controls flower bud formation via blue light-dependent phosphorylation; (2) a combination of a truncated abscisic acid-insensitive 1 (ABI) and pyrabactin-resistant-like (PYL) protein, which can be multimerized by the plant hormone abscisic acid (ABA); or (3) a combination of FKBP and the FRB (FKBP-rapamycin binding) domain in TOR kinase, which can be multimerized by rapamycin and rapamycin analogs; or (4) a combination of GAI (Gibberellin insensitive DWARF1) protein with GID1 (Gibberellin insensitive DWARF1) protein, which can be dimerized by the binding of the plant hormone gibberellin (GA). Examples of combinations with insensitive proteins, etc., may be appropriately provided. The multimerizing sequences used in these combinations are interchangeable. For example, an EV of this disclosure comprising an engineered polypeptide comprising a multimerizing domain containing FKBP and a cargo entity, and an engineered polypeptide comprising an FRB domain and an I-BAR domain, and an EV of this disclosure comprising an engineered polypeptide comprising a multimerizing domain containing FKBP and an I-BAR domain, and an engineered polypeptide comprising an FRB domain and a cargo entity, can be appropriately designed by those skilled in the art as equivalent, non-limiting forms herein. 【0068】In one non-limiting form, this specification provides an engineered polypeptide comprising (a) a polymerizing domain containing an FKBP sequence and (b) a cargo entity. In some embodiments, the engineered polypeptide may further include a linker that connects the polymerizing sequence containing the FKBP sequence and the cargo entity. In some embodiments, the linker may include: (1) an amino acid sequence selected from SEQ ID NOs: 1 to 6; (2) an amino acid sequence exhibiting at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6; or (3) a functional fragment of (1) or (2). 【0069】 In some non-limiting embodiments herein, the multimerization sequence I comprising the FKBP sequence comprises a sequence capable of dimerizing with the FRB sequence. Examples of such non-limiting forms of FKBP sequences include: (1) the amino acid sequence of SEQ ID NO: 9; (2) an amino acid sequence exhibiting at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9 and capable of dimerizing with the FRB sequence; or (3) a functional fragment of (1) or (2). 【0070】In non-limiting embodiments herein, the multimerization sequence II containing the FRB sequence includes a sequence that can form a dimer with the FKBP sequence. Examples of such non-limiting forms of FRB sequences include: (1) the amino acid sequence of SEQ ID NO: 10; (2) an amino acid sequence that exhibits at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10 and can form a dimer with the FKBP sequence; or (3) a functional fragment of (1) or (2). The amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10 are shown in Table 3 below. 【0071】 Whether or not a polymer containing a dimer has been formed can be appropriately confirmed by those skilled in the art using methods known to those skilled in the art, such as ELISA and surface plasmon resonance, to confirm binding activity, as well as by high-throughput methods such as fluorescence resonance energy transfer (FRET) (T Lin et al., Protein Sci. 113, 3114-3119 (2016)) and bioluminescence resonance energy transfer (BRET) (M Zhou et al., ChemMedChem 11, 738-756 (2016)), which detect the proximity of proteins using fluorescence or luminescence, and protein complementation assay (PCA) which utilizes the reconstitution of fluorescent proteins or enzymes using split lucifers, etc. (AS Dixon et al., ACS Chem. Biol. 11, 400-408 (2016) and Y. Matsuyama et al., Anal. Chem. 90, 3001-3004 (2018), etc.). 【0072】(Inducing Factors) Those skilled in the art can appropriately select suitable inducing factors depending on the combination of the multimerized sequence I and the multimerized sequence II. For example, when the combination of multimerized sequence I and multimerized sequence II is the combination of FRB and FKBP, the combination of split FRB (FRB T2098L) and FKBP described later, and the combination of split FKBP and FRB, rapamycin (CAS NO: 53123-88-9) and rapamycin analogs are preferably exemplified as the inducing factors. Some non-limited forms of the rapamycin analog include AP21967, CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap), C16-(S)-butylsulfonamiderapamycin (C16-BSrap), C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry & Biology 13, 99-107 (2006)), AZD8055, BEZ235 (NVP-BEZ235), chrysophanic acid (chrysophanol), defololimus (MK-8669), everolimus (RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (Selleck (Houston, TX, Preferably, examples include those available from the USA. When the combination of multimerization sequence I and multimerization sequence II is a combination of GID1 protein and GAI protein, gibberellins and gibberellin analogs are preferably exemplified as the inducer. As a non-limiting form of the gibberellin analog, GA3-AM containing an acetoxymethyl group is preferably exemplified. When the combination of multimerization sequence I and multimerization sequence II is a combination of a truncated form of abscisic acid insensitivity 1 (ABI) and pyrabactin resistance-like (PYL) protein, abscisic acid and abscisic acid analogs are preferably exemplified as the inducer.As an unspecified form of the abscisic acid analog, (+)-(2Z,4E)-5-(1',4'-Dihydroxy-6',6'-dimethyl-2'-methylenecyclohexyl)-3-methyl-2,4-pentadienoic Acid (Oritani et al., Agric. Biol. Chem., 48, 6, 1677-1678 (1984)) is a suitable example. Furthermore, as the I-BAR domain included in the manipulated polypeptide, one or more I-BAR domains selected from MIM, IRSp53, IRTKS, Pinkbar, and ABBA are preferably used. 【0073】 (I-BAR domain) In some non-limiting embodiments herein, some non-limiting forms of the I-BAR sequence contained in the I-BAR domain include: (1) the amino acid sequence of SEQ ID NO: 11, (2) the amino acid sequence of SEQ ID NO: 12, (3) the amino acid sequence of SEQ ID NO: 13, (4) the amino acid sequence of SEQ ID NO: 14, (5) the amino acid sequence of SEQ ID NO: 15, (6) the amino acid sequence of SEQ ID NO: 16, (7) the amino acid sequence of SEQ ID NO: 17, (8) the amino acid sequence of SEQ ID NO: 18, (9) the amino acid sequence of SEQ ID NO: 19, (10) the amino acid sequence of SEQ ID NO: 20, (11) the amino acid sequence of SEQ ID NO: 21, (12) the amino acid sequence of SEQ ID NO: 22, or (13) Examples of amino acid sequences that exhibit at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with amino acid sequences selected from SEQ ID NOs. 11 to 22 are shown in Table 4 below. 【0074】A person skilled in the art can determine whether an I-BAR domain can form cellular protrusions using known methods such as microscopic observation of cells manipulated to express the I-BAR domain in question. During microscopic observation, a person skilled in the art can further confirm that fluorescent proteins such as GFP are localized to cellular protrusions by detecting trends using a fluorescence microscope on cells that express a modified polypeptide in which a fluorescent protein such as GFP is linked to the I-BAR domain in question. Furthermore, several high-throughput, high-content screening platforms capable of quantifying the formation of cellular protrusions from the aforementioned manipulated cells are available (e.g., HZ Sailem et al., Nat. Commun. 6, 5825 (2015); V Urbanéié et al., J. Cell. Biol. 216, 10, 3405-3422 (2017); G. Jacquemet et al., Methods Mol. Biol. 2040, 359-373 (2019); S Nilfar et al., BMC Sys. Biol. 7, 66 (2013)). Those skilled in the art can use these high-content screening platforms to quantify the formation of cellular protrusions from the aforementioned manipulated cells. 【0075】(Cargo Entities) In some non-limiting embodiments herein, the disclosed technology includes cargo entities. In some embodiments, lipid bilayer particles or a collection of lipid bilayer particles include cargo entities. In some embodiments, the disclosed technology includes one or more cargo entities, such as a plurality of cargo entities (e.g., a first cargo entity, a second cargo entity, or a combination thereof). Cargo entities described herein may be any chemical class, e.g., polypeptides, nucleic acids, sugars, lipids, low molecular weight entities, and combinations thereof. In some non-limiting embodiments herein, the cargo entity is a cargo molecule. In some embodiments, the cargo entity is a viral nucleocapsid or a derivative thereof. In some embodiments, the cargo entity includes viral nucleocapsids, synthetic nucleic acids, transcription factors, recombinases, base editors (base editing factors), prime editors, nucleases (e.g., TALENs, ZFNs, etc.), kinases, kinase inhibitors, receptor signaling activators or inhibitors, intrabodies, chromatin-modifying synthetic transcription factors, innate transcription factors, CRISPR-Cas family proteins, DNA molecules, RNA molecules including sgRNA, or ribonucleoprotein complexes. 【0076】 In some embodiments, the cargo entity is a polymer assembly (e.g., a viral capsid, VLP, viral particle, or functional fragment thereof). 【0077】 In some embodiments, the cargo entity is an intraparticular macromolecules (e.g., a lentiviral core, an AAV particle, another viral core, a VLP core, and its subunits). 【0078】In some embodiments, the cargo entity is a nucleocapsid. In some embodiments, the nucleocapsid is a viral nucleocapsid. In some embodiments, the nucleocapsid is a recombinant viral nucleocapsid. In some embodiments, the nucleocapsid comprises a cargo nucleic acid and a cargo polypeptide. 【0079】 In some embodiments, the cargo entity encodes or encodes an AAV nucleocapsid, an LVV nucleocapsid, or a functional fragment thereof. 【0080】 In some embodiments, the cargo entity may be a cytosol-directed cargo entity or a nuclear-directed cargo entity. For example, in some non-limiting embodiments, the cargo entity may be fused with a nuclear localization sequence (NLS). In other words, in some embodiments, the cargo entity may be a cytoplasmic protein or a cytoplasmic peptide. 【0081】In some embodiments, the cargo entity is a polypeptide cargo entity. In some embodiments, the cargo entity is a cytoplasmic cargo molecule, which can be any peptide, polypeptide, or protein delivered to the target cell, such as an enzyme, a therapeutic agent (e.g., antibody, inhibitor, agonist, and antagonist), or a fluorescent protein. In some embodiments, the cytoplasmic cargo entity can be selected from any one or more of the following: base editors, prime editors, TALENs, ZFNs, kinases, kinase inhibitors, receptor signaling activators or inhibitors, intracellular antibodies, chromatin modification synthesis transcription factors, native transcription factors, and their variants. In some embodiments, the cytoplasmic cargo entity is a CRISPR enzyme, e.g., a type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme is a Cas9 protein (e.g., not only native bacterial Cas9, but any chimera, variant, homolog, or homolog). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also called Csnl and Csxl2), Cas10, Cas11, Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Examples include Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, their homologs, or modified variants thereof. 【0082】In some embodiments, the cargo entity is a nucleic acid cargo entity. In some embodiments, the cargo entity may be a nucleic acid (e.g., DNA or RNA) or another entity. Nucleic acid cargo entities include, but are not limited to, DNA, mRNA encoding the protein or peptide of interest, as well as siRNA, shRNA, miRNA, antisense oligonucleotides, sgRNA, and combinations thereof that target the protein, peptide, or DNA of interest. Other potential cargo entities include, but are not limited to, viral and nonviral vectors expressed intracellularly (or deliverable into the cytoplasm of a cell), ribonucleoprotein complexes such as CRISPR-type entities, and endogenous complexes such as DICER or RISC bound to native or synthetic RNA such as miRNA, shRNA, etc. In fact, those skilled in the art will recognize that any entity that can be expressed intracellularly or physically delivered into the intracellular space can be fused (genetically or synthetically) to a multimerization sequence I, which is explicitly considered herein. Genetic fusion can be achieved by expressing two or more components of a manipulated polypeptide in a producing cell, such as lipid bilayer particles (e.g., cell membrane-derived vesicles, particularly cell protrusion-derived particles). 【0083】In this specification, as an unspecified embodiment where the cargo entity is a nucleic acid cargo entity such as RNA, a form in which the nucleic acid cargo entity is indirectly bound to an engineered polypeptide containing a multimerization sequence I and a nucleic acid-binding polypeptide via the nucleic acid-binding polypeptide may also be exemplified. Suitable examples of such unspecified combinations of nucleic acid-binding polypeptides and nucleic acids capable of binding to them include the MS2 bacteriophage protein and an RNA molecule linked to one or more WTLs (wild-type loops) or high-affinity loops (HALs) capable of binding to MS2 (ME Hung et al., J. Extracell. Vesicles., 5, 31027 (2016)). Another unspecified combination of nucleic acid-binding polypeptides and nucleic acids capable of binding to them also includes the retrovirus structural core protein Gag and an RNA molecule (D. Mazurov et al., Viruses, 15, 690, v15030690 (2023)). Similarly, Nano MEDIC technology (P Gee et al., Nat Comm. 11, 1334, s41467-020-14957-y (2020)) is also preferably exemplified as another non-limiting combination of nucleic acid-binding polypeptide and nucleic acid capable of binding to said polypeptide. A non-limiting form of using such Nano MEDIC technology is preferably exemplified by a combination of an engineered polypeptide expressed from recombinant nucleic acid containing an sgRNA flanked by self-cleaving ribozyme sequences such as HH and HDV upstream of the cDNA encoding the HIV retroviral Gag protein, and further ligated an extended packaging signal Psi to a multimerization sequence I such as FRB upstream of the sgRNA, and an engineered polypeptide containing a multimerization sequence II such as FKBP1 and an I-BAR domain that can associate with multimerization sequence I such as FRB. 【0084】In addition, in any of the embodiments described above, an engineered polypeptide comprising a second cargo entity is provided. Membrane-bound cargo entities are preferably exemplified as such a second cargo entity. In some non-limiting embodiments, the membrane-bound cargo entity may comprise (i) a targeted peptide / protein and (ii) a transmembrane domain. Exemplary targeted peptides include, but are not limited to, any antibody or antigen-binding fragments such as Fab, Fab', and F(ab')2, Fd, scFv, single-chain antibodies, disulfide-bonded Fvs(sdFv), and nanobodies. The targeted peptide (e.g., scFv) can bind to a target of interest on a specific cell type such as a T cell. In some non-limiting embodiments, the targeted peptide may be Fab, Fab', and F(ab')2, Fd, scFv, single-chain antibodies, disulfide-bonded Fvs(sdFv), de novo-designed binding molecules, afibodies, DARPIN, or nanobodies. 【0085】 Transmembrane domains (TMDs) are known in the art. Transmembrane domains (TMDs) consist mainly of nonpolar amino acid residues and span the bilayer once or multiple times. TMDs typically consist of an α-helix structure. Peptide bonds are polar and can include internal hydrogen bonds formed between a carbonyl oxygen atom and a hydrateable amide nitrogen atom. Within lipid bilayers where water is substantially excluded, peptides typically adopt an α-helix configuration to maximize internal hydrogen bonding. Helix lengths of 18 to 21 amino acid residues are usually sufficient to span the typical width of the lipid bilayer. TMDs oriented at the extracytoplasmic N-terminus and cytoplasmic C-terminus are classified as Type I TMDs, and TMDs oriented at the extracytoplasmic C-terminus and cytoplasmic N-terminus are classified as Type II TMDs. In some non-limiting embodiments of the disclosed extracytoplasmic domains, they are classified as Type I and as Type II if they are intracytoplasmic. In some non-limiting embodiments, the transmembrane domain is a single-pass type I transmembrane domain containing 18–21 amino acids, where at least about 90% of the amino acids are nonpolar. 【0086】For example, non-limiting examples of TMDs suitably usable with the manipulated polypeptide include transmembrane domains of cell receptors, such as the platelet-derived growth factor receptor (PDGFR) transmembrane domain represented by SEQ ID NO: 23 (AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR), which can be encoded by the nucleotide sequence of SEQ ID NO: 24 (GCCGTCGGCCAGGACACCCAAGAAGTGATCGTCGTCCCTCACAGCCTGCCTTTCAAGGTGGTGGTCATCAGCGCCATTCTGGCCCTGGTGGTGCTGACCATCATCAGCCTGATCATCCTGATTATGCTGTGGCAGAAGAAGCCCAGA). The TMD may be directly linked to the target peptide (e.g., scFv), or it may be linked via a linker. In some embodiments, the linker connecting the TMD to the target peptide / protein comprises an amino acid sequence of (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more. In some embodiments, the linker connecting the TMD and the target peptide includes (1) an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or (2) an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6. 【0087】 As another non-limiting form of the second cargo entity, a preferred example is one in which sgRNA is contained within a lipid bilayer particle as the second cargo entity when the first cargo entity is a CRISPR enzyme such as the Cas9 protein. 【0088】 To produce lipid bilayer particles containing a second cargo entity together with a first cargo entity, several known techniques can be appropriately utilized by those skilled in the art. For example, one possible form is to prepare each manipulated polypeptide using different sequences as the multimerization sequence I contained in the manipulated polypeptide containing the first cargo entity and the multimerization sequence I contained in the manipulated polypeptide containing the second cargo entity. For such an purpose, polynucleotides encoding manipulated polypeptides containing different multimerization sequences I can be produced, and then polynucleotides encoding manipulated polypeptides containing a multimerization sequence II and an I-BAR domain that can associate with each multimerization sequence I can be produced. Host cells transformed with vectors containing each manipulated polynucleotide can then be cultured to isolate lipid bilayer particles containing both the first and second cargo entities. In this case, the I-BAR domains in the manipulated polypeptide containing the multimerization sequence II that can associate with each multimerization sequence I may be the same (e.g., MIMs together), or they may be different (e.g., a combination of MIM and Pinkbar). 【0089】As another non-limiting embodiment for producing lipid bilayer particles containing a second cargo entity together with a first cargo entity, a preferred example is a method for producing manipulated polypeptides containing each split sequence, the second cargo entity, and the first cargo entity separately, using split sequences of those sequences as multimerization sequence I or multimerization sequence II that can associate with multimerization sequence I. As such non-limiting embodiments, those skilled in the art can appropriately utilize known techniques using split FRB (FRB T2098L) or split FKBP (HD Wu et al., Nat. Methods, 17, 9, 928-936 (2020)). As one non-limiting form of using known techniques that employ such a split FRB (FRB T2098L) or split FKBP, for example, upstream of the cDNA encoding the Gag protein of the HIV retroviral, an sgRNA flanked by self-cleaving ribozyme sequences such as HH and HDV is included, and further upstream of the sgRNA, an extended packaging signal Psi is included, and sFKBP is included. 1N Recombinant nucleic acids linked to a multimerized sequence I' such as split-type FKBP1, and sFKBP 1C Recombinant nucleic acids linked to split-type multimerized sequences I'' such as split-type FKBP1, two modified polypeptides expressed from these nucleic acids, and sFKBP 1N Split-type multimerized sequences I' and sFKBP 1C Combinations of a modified polypeptide containing an I-BAR domain and a multimerizing sequence II such as FRB, which can associate with a split-type multimerizing sequence I'' to form a trimer, are also preferably exemplified. 【0090】 (Manipulated Lipid Bilayer Particles) The Disclosure also provides manipulated lipid bilayer particles and preparations thereof. The Disclosure also provides manipulated extracellular microparticles and preparations thereof. In another non-limiting embodiment, the Disclosure provides manipulated cell membrane-derived vesicles. Furthermore, in another non-limiting embodiment, the Disclosure provides manipulated filopodium-derived vesicles (FDVs). 【0091】In this specification, the disclosure provides engineered lipid bilayer particles comprising a combination of engineered polypeptides from any of the embodiments described above. Non-limiting embodiments of such engineered lipid bilayer particles include engineered lipid bilayer particles comprising one or more combinations of a first engineered polypeptide comprising a multimerization sequence I and a cargo entity, and an engineered polypeptide comprising a second multimerization sequence II and an I-BAR (Inverse-Bin / Amphiphysin / Rvs) domain that can associate with multimerization sequence I. Non-limiting embodiments of such lipid bilayer particles also preferably include engineered extracellular microparticles, engineered cell membrane-derived vesicles, and engineered cell protrusion-derived particles. 【0092】 As described above, any combination of manipulated polypeptides in the manipulated lipid bilayer particles forms a multimer containing cargo entities through the binding of multimerization sequence I and multimerization sequence II. While not intended to be constrained by any particular theory, the localization of this multimer to the cell membrane or cellular protrusions allows the manipulated lipid bilayer particles to contain a high concentration of the desired cargo entities with minimal contamination from impurities and denatured proteins. Furthermore, these lipid bilayer particles exhibit low immunogenicity derived from viral foreign proteins. Therefore, the desired cargo entities can be efficiently delivered to cells by lipid bilayer particles with low immunogenicity. 【0093】Some non-limiting forms of the combination of multimerization sequence I and multimerization sequence II contained in some of the manipulated lipid bilayer particles include: (1) a combination of the photoreceptor cryptochrome 2 (CRY2), which is induced to multimerize by blue light, and the CRY-interacting basic helix-loop-helix 1 (CIB1) protein, which controls flower bud formation via blue light-dependent phosphorylation; (2) a combination of a truncated abscisic acid-insensitive 1 (ABI) and pyrabactin-resistant-like (PYL) protein, which is induced to multimerize by the plant hormone abscisic acid (ABA); or (3) a combination of FKBP and the FRB (FKBP-rapamycin binding) domain in TOR kinase, which is induced to multimerize by rapamycin and rapamycin analogs; or (4) a combination of GAI (Gibberellin insensitive DWARF1) protein with GID1 (Gibberellin insensitive DWARF1) protein, which is induced to dimerize by the binding of the plant hormone gibberellin (GA). Examples of combinations with insensitive proteins, etc., can be appropriately illustrated. The multimerizing sequences used in these combinations are interchangeable. For example, a lipid bilayer particle comprising an engineered polypeptide comprising a multimerizing domain containing FKBP and a cargo entity, and an engineered polypeptide comprising an FRB domain and an I-BAR domain, as well as an engineered lipid bilayer particle comprising a combination of an engineered polypeptide comprising a multimerizing domain containing FKBP and an I-BAR domain, and an engineered polypeptide comprising an FRB domain and a cargo entity, can be appropriately designed by a person skilled in the art as an equivalent, non-limiting form as described herein. Furthermore, as described above, an engineered lipid bilayer particle comprising an engineered polypeptide comprising two types of split-type FKBP and two types of split-type FRB, respectively, and an engineered polypeptide comprising FRB and FKBP that can associate with each other (form trimers), can also be appropriately designed by a person skilled in the art as an equivalent, non-limiting form as described herein. As an unspecified form of the lipid bilayer particle, examples of the lipid bilayer particle include engineered extracellular microparticles, engineered cell membrane-derived vesicles, and engineered cell protrusion-derived particles.Furthermore, as the I-BAR domain included in the manipulated polypeptide, one or more I-BAR domains selected from MIM, IRSp53, IRTKS, Pinkbar, and ABBA are preferably used. 【0094】An engineered extracellular particle comprising one or more combinations of the engineered polypeptides of any of the embodiments described above may optionally contain an inducer for the multimerization of multimerization sequence I and multimerization sequence II contained in the engineered polypeptide. The inducer can be appropriately selected by those skilled in the art depending on the combination of multimerization sequence I and multimerization sequence II. For example, when the combination of multimerization sequence I and multimerization sequence II is a combination of FRB and FKBP, a combination of split FRB (FRB T2098L) and FKBP, and a combination of split FKBP and FRB, rapamycin (CAS NO: 53123-88-9) or a rapamycin analog is preferably exemplified as the inducer. Some non-limited forms of the rapamycin analog include AP21967, CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap), C16-(S)-butylsulfonamiderapamycin (C16-BSrap), C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry & Biology 13, 99-107 (2006)), AZD8055, BEZ235 (NVP-BEZ235), chrysophanic acid (chrysophanol), defololimus (MK-8669), everolimus (RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (Selleck (Houston, TX, Preferably, examples include those available from the USA. When the combination of multimerization sequence I and multimerization sequence II is a combination of GID1 protein and GAI protein, gibberellins and gibberellin analogs are preferably exemplified as the inducer. As a non-limiting form of the gibberellin analog, GA3-AM containing an acetoxymethyl group is preferably exemplified. When the combination of multimerization sequence I and multimerization sequence II is a combination of a truncated form of abscisic acid insensitivity 1 (ABI) and pyrabactin resistance-like (PYL) protein, abscisic acid and abscisic acid analogs are preferably exemplified as the inducer.As an unspecified form of the abscisic acid analog, (+)-(2Z,4E)-5-(1',4'-Dihydroxy-6',6'-dimethyl-2'-methylenecyclohexyl)-3-methyl-2,4-pentadienoic Acid (Oritani et al., Agric. Biol. Chem., 48, 6, 1677-1678 (1984)) is a suitable example. Furthermore, as the I-BAR domain included in the manipulated polypeptide, one or more I-BAR domains selected from MIM, IRSp53, IRTKS, Pinkbar, and ABBA are preferably used. 【0095】 (Manipulated Polynucleotides) One or more combinations of manipulated polynucleotides (recombinant nucleic acids) encoding manipulated polypeptides comprising a multimerizing sequence I and a cargo entity, as described in some of the above-described non-limiting embodiments, and manipulated polypeptides comprising a multimerizing sequence II and an I-BAR (Inverse-Bin / Amphiphysin / Rvs) domain that can associate with multimerizing sequence I, are also disclosed as one of the other non-limiting embodiments herein. 【0096】Preferred examples of polynucleotides encoding a multimerizing sequence contained in the polynucleotide encoding an engineered polypeptide disclosed herein include: (a) the polynucleotide sequence represented by SEQ ID NO: 25; (b) a polynucleotide variant exhibiting at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polynucleotide sequence represented by SEQ ID NO: 10 (FRB sequence); or (c) a functional fragment of (a) or (b). 【0097】 Preferred examples of polynucleotides encoding a multimerizing sequence contained in the polynucleotide encoding an engineered polypeptide disclosed herein include: (a) the polynucleotide sequence represented by SEQ ID NO: 26; (b) a polynucleotide variant exhibiting at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polynucleotide sequence represented by SEQ ID NO: 9 (FKBP1 sequence); or (c) a functional fragment of (a) or (b). The polynucleotide sequences of sequence numbers 25 and 26 are shown in Table 5 below. 【0098】 As an unspecified form of a polynucleotide encoding an I-BAR (Inverse-Bin / Amphiphysin / Rvs) domain contained in a polynucleotide encoding an engineered polypeptide disclosed herein; (1) the polynucleotide sequence represented by SEQ ID NO: 27, (2) the polynucleotide sequence represented by SEQ ID NO: 28, (3) the polynucleotide sequence represented by SEQ ID NO: 29, (4) the polynucleotide sequence represented by SEQ ID NO: 30, (5) the polynucleotide sequence represented by SEQ ID NO: 31 (6) A polynucleotide variant that exhibits at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a polynucleotide sequence selected from SEQ ID NOs: 27 to 31, and whose encoded polypeptide is capable of forming cellular protrusions; or (7) Any functional fragment of (1) to (6); are preferably exemplified. The polynucleotide sequences of SEQ ID NOs: 27 to 31 are shown in Table 6 below. 【0099】In one non-limiting form, this specification provides a first engineered polynucleotide encoding a multimerization sequence I and a cargo entity, and a second engineered polynucleotide encoding a multimerization sequence II and an I-BAR (Inverse-Bin / Amphiphysin / Rvs) domain that can associate with multimerization sequence I. In some embodiments, the engineered polynucleotide sequence may further include a linker that ligates multimerization sequence I and the cargo entity, and a linker that ligates multimerization sequence II and the I-BAR (Inverse-Bin / Amphiphysin / Rvs) domain. In some embodiments, the linker may include: (1) an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; (2) an amino acid sequence exhibiting at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6; or (3) a functional fragment of (1) or (2). 【0100】Vectors comprising one or more combinations of the manipulated polynucleotides described herein are also suitably exemplified as non-limiting embodiments. Examples of non-limiting embodiments of the vectors disclosed herein include eukaryotic expression vectors comprising a heterogeneous promoter operably linked to the manipulated polynucleotides (e.g., SV40 promoters such as the SV40 early promoter and SV40 late promoter, CMV promoters such as the human cytomegalovirus promoter, or retroviral promoters such as the mouse mammary cancer virus promoter and the Rous sarcoma virus promoter, or inducible promoters such as the metallothionein promoter), as well as a regulatory sequence comprising an enhancer and a cis-acting element including a polyA sequence, and prokaryotic expression vectors comprising a constitutive or inducible promoter (e.g., T3, T7, Lac, trp, phoA), a ribosome binding site, or a transcription terminator. For example, when the prokaryotic host is Escherichia coli such as JM109, DH5α, HB101, or XL1-Blue, vectors containing promoters that can be efficiently expressed in Escherichia coli, such as the lacZ promoter (Wardet al., Nature, 341, 544-546 (1989); FASEB J., (1992) 6, 2422-2427), the araB promoter (Betteret al., Science, 240, 1041-1043 (1988)), or the T7 promoter, are preferably exemplified. Examples of such vectors, in addition to the above vectors, include pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, or pET (in this case, BL21, which expresses T7 RNA polymerase, is preferred as the host).Suitable vectors for using eukaryotes as host cells include, for example, mammalian expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nuc. Acids. Res., 18, 17, 5322 (1990)), pEF, pCDM8), insect cell expression vectors (e.g., "Bac-to-BAC baculovairus expression system" (GIBCO BRL), pBacPAK8), plant expression vectors (e.g., pMH1, pMH2), animal virus expression vectors (e.g., pHSV, pMV, pAdexLcw), retrovirus expression vectors (e.g., pZIPneo), and yeast expression vectors (e.g., "Pichia Expression Kit" (Invitrogen)). 【0101】 When the goal is to express a gene in animal cells such as CHO cells, COS cells, NIH3T3 cells, HEK293, HEK293FS (Free Style), mesenchymal stem cells, megakaryocytes, induced pluripotent stem cells (iPSCs), T cells, erythrocytes, erythrocyte precursors, and iPSC-derived versions of any of the aforementioned cells, it is essential that the gene possesses the necessary promoters for intracellular expression, such as the SV40 promoter (Mulliganet al., Nature, 277, 108 (1979)), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nuc. Acids Res., (1990) 18, 5322), CAG promoter (Gene, (1991) 108, 193), and CMV promoter. It is even more preferable if the gene has genes for selecting transformed cells. Examples of genes for selecting transformed cells include drug resistance genes that can be identified by drugs (such as neomycin and G418). Examples of vectors with such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13. 【0102】Furthermore, for stable gene expression and intracellular copy number amplification, one method involves introducing a vector containing the complementary DHFR gene (e.g., pCHOI) into CHO cells lacking the nucleic acid synthesis pathway, and amplifying the result with methotrexate (MTX). For transient gene expression, another method involves transforming COS cells, which have a gene expressing the SV40 T antigen on their chromosome, with a vector containing an SV40 replication origin (e.g., pcD). Replication origins can also be derived from polyomaviruses, adenoviruses, bovine papillomavirus (BPV), etc. In addition, expression vectors can include aminoglycoside transferase (APH) genes, thymidine kinase (TK) genes, Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) genes, dihydrofolate reductase (dhfr) genes, etc., as selection markers for gene copy number amplification in the host cell line. 【0103】(Method for Loading Cargo Entities) In another aspect of this specification, a method for loading cargo entities into lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles) such as extracellular vesicles (EVs) is also provided. A method for loading such cargo entities into lipid bilayer particles (e.g., cell membrane-derived vesicles, cell protrusion-derived particles) includes expressing the engineered polypeptide of the above embodiments in a host cell. For example, a method for loading cargo entities into lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles) preferably includes: (1) in a eukaryotic host cell, DNA or RNA encoding an engineered polypeptide containing cargo entities; and (2) in a eukaryotic host cell, DNA or RNA expressing an engineered polypeptide containing a recombinant I-BAR domain. The DNA or RNA of the engineered polypeptide containing cargo entities may be expressed from an expression vector transfected into a host cell suitable for producing the disclosed lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles). The host vector may be transfected transiently or stably. The DNA or RNA of (1) and (2) may be contained in a single expression vector, or the DNA or RNA of (1) and (2) may be contained in separate expression vectors. 【0104】To facilitate the loading of cargo entities into lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles) during the expression of the polypeptide, the method may include contacting the host cell expressing the polypeptide with an inducing factor that induces association between a multimerization sequence I contained in the engineered polypeptide containing cargo entities and a multimerization sequence II contained in the engineered polypeptide containing a recombinant I-BAR domain. Those skilled in the art can appropriately select an appropriate inducing factor depending on the type of combination of multimerization sequences I and II. Suitable examples of such inducing factors include rapamycin and rapamycin analogs, gibberellins and gibberellin analogs, abscisic acid and abscisic acid analogs, and blue light irradiation. Furthermore, as the I-BAR domain contained in the engineered polypeptide, one or more I-BAR domains selected from MIM, IRSp53, IRTKS, Pinkbar, and ABBA are preferably used. 【0105】In some non-limiting embodiments herein, the cargo entity may be an enzyme, a therapeutic agent (e.g., an antibody, inhibitor, agonist, and antagonist), or any polypeptide of interest to be loaded into a target cell, such as a fluorescent protein. In some non-limiting embodiments, the cargo entity may be a base editor, a prime editor, a TALEN, a ZFN, a kinase, a kinase inhibitor, a receptor signaling factor, an intron, a chromatin modification synthesis transcription factor, a native transcription factor, or a variant thereof. In some other non-limiting embodiments, the cargo entity may be a CRISPR enzyme, e.g., a type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme may be any Cas9 protein, e.g., any naturally occurring bacterial Cas9, as well as any chimera, mutant, homolog, or orthologue. A method of loading sgRNA or the like as a second cargo entity into lipid bilayer particles (e.g., cell membrane-derived vesicles, cell protrusion-derived particles) along with the CRISPR enzyme is also exemplified as an unspecified, non-limiting embodiment. 【0106】 Nucleic acids (e.g., DNA or RNA) are also preferably exemplified as one of several non-limiting embodiments of cargo entities loaded into lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles). Nucleic acid cargo entities may include, but are not limited to, DNA encoding the target protein or peptide, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, and combinations thereof. Other non-limiting forms of cargo entities may include, but are not limited to, viral and non-viral vectors expressed intracellularly (or delivered to the cellular cytosol), or ribonucleoprotein complexes such as CRISPR-type entities and endogenous complexes bound to native or synthetic RNA. 【0107】(Method for producing lipid bilayer particles containing cargo entities) Another aspect of this specification also provides a method for producing lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles) such as extracellular vesicles (EVs) containing cargo entities. A method for producing such lipid bilayer particles containing cargo entities (e.g., cell membrane-derived vesicles, cell protrusion-derived particles) includes a method for recovering lipid bilayer particles containing cargo entities (e.g., cell membrane-derived vesicles, cell protrusion-derived particles) from the culture supernatant of host cells expressing the manipulated polypeptides described in the above embodiments. For example, a method for producing lipid bilayer particles containing cargo entities (e.g., cell membrane-derived vesicles or cell protrusion-derived particles) is preferably exemplified as one non-limiting embodiment: (1) culturing host cells expressing a first engineered polypeptide containing a multimerizing sequence I and cargo entities, and a second engineered polypeptide containing a multimerizing sequence II and an I-BAR domain that can associate with the multimerizing sequence I, in a culture medium in the presence of a multimerization inducer that can induce the association of the multimerizing sequence I and the multimerizing sequence II; and (2) collecting the culture supernatant from the culture medium described in (1). A preferably exemplified method is one comprising expressing a first engineered polypeptide containing a multimerizing sequence I and cargo entities, and a second engineered polypeptide containing a multimerizing sequence II and an I-BAR domain that can associate with the multimerizing sequence I. The DNA or RNA of the engineered polypeptide containing cargo entities can be expressed from an expression vector transfected into a host cell suitable for producing the disclosed lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles). The host vector may be transfected transiently or stably. The DNA or RNA of (1) and (2) may be contained in a single expression vector, or the DNA or RNA of (1) and (2) may be contained in separate expression vectors. 【0108】To facilitate the loading of cargo entities into lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles) during the expression of the polypeptide, the method may include contacting the host cell expressing the polypeptide with an inducing factor that induces association between a multimerization sequence I contained in the engineered polypeptide containing cargo entities and a multimerization sequence II contained in the engineered polypeptide containing a recombinant I-BAR domain. Those skilled in the art can appropriately select an appropriate inducing factor depending on the type of combination of multimerization sequences I and II. Suitable examples of such inducing factors include rapamycin and rapamycin analogs, gibberellins and gibberellin analogs, abscisic acid and abscisic acid analogs, and blue light irradiation. Furthermore, as the I-BAR domain contained in the engineered polypeptide, one or more I-BAR domains selected from MIM, IRSp53, IRTKS, Pinkbar, and ABBA are preferably used. 【0109】 Furthermore, in an unspecified form, host cells expressing an engineered polypeptide comprising a multimerizing sequence I and a cargo entity, and an engineered polypeptide comprising a multimerizing sequence II and an I-BAR domain that can associate with said multimerizing sequence I, are cultured in a culture medium suitable for expressing the engineered polypeptide in the host cells by static culture, agitation culture (using a stirrer or airlift type), or shaking culture (reciprocating shaking type or rotational shaking type), thereby releasing lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles). Methods well known to those skilled in the art are used for recovering the lipid bilayer particles (e.g., cell membrane-derived vesicles or cell protrusion-derived particles) released from the host cells in various volumes ranging from small to large, and for purifying the recovered solution. As an unrestricted form of such well-known methods, precipitation methods using crowding reagents such as polyethylene glycol, centrifugation (any centrifugation method such as high-speed ultracentrifugation or low-speed centrifugation), cross-flow filtration, column chromatography, affinity purification, etc., may be used as appropriate. 【0110】 (Pharmaceuticals containing lipid bilayer particles with cargo entities) The lipid bilayer particles containing cargo entities provided herein can be formulated as pharmaceutical compositions in ways well known to those skilled in the art. For example, they can be used parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids, or as injectable suspensions. For example, they can be formulated by mixing them with pharmacologically acceptable carriers or media, specifically sterile water or saline solution, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., in a unit dose form generally required for pharmaceutical practice. The amount of active ingredient in these formulations is set so as to obtain an appropriate volume within the indicated range. 【0111】 Sterile compositions for injection can be formulated in accordance with standard formulation practices using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, glucose, and isotonic solutions containing other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable solubilizers, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80™, HCO-50, etc.), may be used in combination. Examples of oily solutions include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may be used as solubilizers. Buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may also be added. The prepared injection solution is usually filled into a suitable ampoule. 【0112】 The pharmaceutical compositions provided herein are preferably administered by parenteral administration. For example, they may be in the form of injections, nasal administrations, pulmonary administrations, or transdermal administrations. For example, they may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. 【0113】The method of administration can be appropriately selected depending on the patient's age and symptoms. The dosage of the pharmaceutical composition containing the antibody or polynucleotide encoding the antibody can be set to an appropriate dose within the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, an appropriate dose within the range of 0.001 to 100,000 mg per patient may be used, but this specification is not necessarily limited to these values. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but a person skilled in the art can set an appropriate dosage and method of administration considering these conditions. 【0114】 (Wound treatment agents, methods for treating wounds, uses for treating wounds, and uses for the manufacture of wound treatment agents) In one non-limiting form of this specification, wound treatment agents comprising engineered lipid bilayer particles containing the cargo entity provided herein as an active ingredient are preferably exemplified. In another different non-limiting form, a method for treating a wound comprising administering the cargo entity provided herein to a subject of interest is also preferably exemplified. In yet another different non-limiting form, the use of engineered lipid bilayer particles containing the cargo entity provided herein for the treatment of wounds is also preferably exemplified. In yet another different non-limiting form, the use of engineered lipid bilayer particles containing the cargo entity provided herein for the manufacture of wound treatment agents is also preferably exemplified. In each of the above forms, engineered lipid bilayer particles containing Rac1 as the cargo entity are preferably exemplified as an active ingredient. 【0115】 The embodiments will be described in more detail below with reference to examples, but these examples do not limit the present disclosure in any way. 【0116】(Materials and Methods) HEK293 cells, available as FreeStyle® 293 cells (Thermo Fisher Scientific, #R79007) adapted for serum-free suspension culture, were used for EV production. The cells were cultured in synthetic FreeStyle 293 Expression Medium (Thermo Fisher Scientific, #12338018) in an orbital shaker rotating at 135 rpm and placed in an incubator at 37°C in a humidified atmosphere of 8% CO2. For EV reception, PANC-1 cells (ATCC, #CRL-1469), HEK293 cells (Lenti-X, TaKaRa, #632164), HeLa cells (ATCC, #CCL-2), and Ca9-22 cells (Japanese Collection of Research Bioresources Cell Bank, #JCRB0625) were cultured at 37°C in a humidified atmosphere of 5% CO2 using Dulbecco's modified Eagle medium (DMEM) (Nacalai, #08459-64) supplemented with 10% (vol / vol) FBS (Sigma, #173012) and penicillin-streptomycin. 【0117】 Human CIP4 cDNA (Gene Bank NC_000019.10; NCBI protein database code gi:62897779) from the pEGFP-N3 plasmid (A. Shimada et al., Cell 129, 761-772 (2007)) was subcloned into the pMXs-IRES-hygromycin-resistant (HygR) plasmid (P. Puzar (z is a character with a Harchek mark) Dominkus (s is a character with a Harchek mark) et al., Biochim Biophys Acta Biomembr 1860, 1350-1361 (2018)) which has mEos4b or mCherry at its C-terminus. 【0118】The I-BAR domain (residues 1-275) of mouse MIM cDNA (Gene Bank XM_006520714.2) was inserted between the BglII and BamHI sites of the pEGFP-C1 plasmid (CLONTECH). EGFP in pEGFP-C1 was replaced with Halo7 or mCherry to create pHalo7-C1 or pmCherry-C1, respectively. Next, the I-BAR domain of MIM was inserted into the pHalo7-C1 or pmCherry-C1 plasmid at the same site as the pEGFP-C1-MIM I-BAR. FKBP cDNA (T. Komatsu et al., Nat Methods 7, 206-208 (2010)) was inserted into the pmCherry-MIM I-BAR to construct pmCherry-FKBP-MIM I-BAR, and mCherry was replaced with YFP to construct pYFP-FKBP-MIM I-BAR. 【0119】 Mouse Rac1 cDNA (Gene Bank NC_000071.7) was inserted into the multi-cloning site of pHalo7-C1 to create pHalo-C1-Rac1. Point mutations of G12V or T17N were introduced into Rac1 using the PrimeSTAR® Mutagenesis Basal Kit (TaKaRa, #R046A). pEGFP-C1-GST-Rac1 was created by inserting glutathione S-transferase (GST)-tagged Rac1 cDNA into the pEGFP-C1 plasmid, as previously reported (WNI Wan Mohamad Noor et al., Sci Adv 9, eadf5143 (2023)). 【0120】Cas12f was expressed in lentivirus using a pKLV2 vector. The puromycin resistance gene and BFP of pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene plasmid #67974) (K. Tzelepis et al., Cell Rep 17, 1193-1205 (2016)) were replaced with IRES-HygR and EGFP, respectively, to form the vector backbone. The I123H / D195K / D208R / V232A mutant of Cas12f derived from Acidibacillus sulfuroxidans (referred to as "Cas12f" in this example) (T. Hino et al., Cell 186, 4920-4935.e4923 (2023)), a Halo7 tag, and FRB were inserted after the CMV promoter of the vector, resulting in pKLV2-AsCas12fHKRA-Halo7-GSGS-FRB-guideRNA. A Cas12f reporter with TTR sgRNA was also constructed under the CMV promoter of the pKLV2 vector. This vector was pKLV2mCherry_TTR_GFPGFPv3, with mCherry followed by T2A and TTR target sequences, then two frameshifted GFP sequences, IRES, and a hygromycin resistance gene. 【0121】PMCherry-FKBP-MIM I-BAR, pEYFP-FKBP-MIM I-BAR, and pKLV2-AsCas12fHKRA-Halo7-GSGS-FRB-guideRNA were prepared using polyethyleneimine MAX (PEI, Polysciences, #49553-93-7) by transfecting HEK293 cells with purified DNA containing pEGFP-C1, pEGFP-N3-MIM I-BAR, pHalo7-C1, pHalo7-C1-MIM I-BAR, pEGFP-C1-GST-Rac1, pHalo7-C1-Rac1 WT, and mutants (G12V and T17N). Plasmid DNA (30 μg) and 1 mg / ml PEI (30 μl) were each diluted in 1 ml of Opti-MEM® reduced serum medium (Thermo Fisher Scientific, #31985070) and incubated at room temperature for 5 minutes. In the case of co-transduction, 15 μg each of the two plasmids were pre-mixed before dilution in Opti-MEM. Next, the DNA and PEI in Opti-MEM were mixed and incubated for a further 30 minutes at room temperature, after which 1 × 10⁶ of this mixture was added to 28 ml of HEK293 cell culture medium. 6 The solution was added to a concentration of cells / ml. The cells were then cultured for 48 hours. The transfected cells were then treated with 10 nM rapamycin or 0.01% DMSO for 24 hours before EV preparation for chemical dimerization. 【0122】Preparation of Knockout and Stable Expression Cell Lines: The CIP4 gene in PANC-1 cells was knocked out using the CRISPR-Cas9 system. Two pairs of guide RNAs targeting the second exon sequence of human CIP4 (#1: 5'-TGCTCGAGCGCCACACGCAG-3' (SEQ ID NO: 32), #2: 5'-CGTGAAAGAACGCACCGAAG-3' (SEQ ID NO: 33)) were designed using the CRISPR Direct Server (https: / / crispr.dbcls.jp / ) (G. Hoxhaj, K. Dissanayake, C. MacKintosh, PLoS One 8, e73327 (2013)). Forward and reverse oligoDNA of these sequences were annealed and inserted into the BbsI site of the pX459 vector (Addgene #62988). PANC-1 cells were transfected with these plasmids and selected with puromycin (InvivoGen, ant-pr-1). 【0123】 The CIP4 gene was reexpressed in CIP4 KO PANC-1 cells by retrovirus-mediated gene transfer. HEK293 platA cells were transfected with pMXs-CIP4-mEos4b-IRES-HygR or pMXs-CIP4-mCherry-IRES-HygR for 2 days using 293-fectin (Invitrogen) to produce retroviruses. The supernatant containing the retroviruses was filtered and used to infect CIP4 KO PANC-1 cells in the presence of 32 μg / ml polyblen (Sigma, TR-1003). Cells were selected using a culture medium containing hygromycin B (InvivoGen, ant-hg-1) to obtain stable transfectants at appropriate concentrations. 【0124】The following antibodies were used for antibody Western blotting (WB) and / or immunofluorescence staining (IF) at the following dilution ratios. Mouse anti-CIP4 (BD, clone 21, 612556, 1:100 for IF, 1:1000 for WB), mouse anti-GAPDH (Santa Cruz, sc-166574, 1:2000 for WB), rabbit anti-WAVE2 (Cell signaling, clone D2C8, #3659, 1:100 for IF, 1:1000 for WB), rabbit anti-N-WASP (Cell signaling, clone 30D10, #4848, 1:1000 for WB), mouse anti-Dynamin II (BD, #610263, 1:1000 for WB), mouse anti-endophyllin II (Santa Cruz, # sc-365704, 1:1000 for WB), rabbit anti-clathrin light chain A (CLTA) (proteintech, #10852-1-AP, Mouse anti-Rac1 (Millipore, 05-389, 1:4000 for WB), Mouse anti-Rac1 (BD, # 610651, 1:200 for EV incorporation WB, 1:400 for ELISA), Rabbit anti-Cdc42 (Cell signaling, # 2462, 1:1000 for WB), Rabbit anti-HaloTag (Promega, # G9281, 1:1000 for WB, 1:1000 for IF, 1:1000 for ELISA), Mouse anti-HaloTag (Promega, # G9211, 1:1000 for WB, 1:1000 for ELISA), Mouse monoclonal anti-MIM (Abnova, # H00009788-M01, 1:1000 for WB), Mouse anti-CD81 (Santa Cruz, sc-166029, 1:2000 for WB), mouse anti-annexin I (Santa Cruz, sc-12740, 1:1000 for WB), mouse anti-β-actin (Santa Cruz, sc-47776, 1:3000 for WB), rabbit anti-GFP (MBL Life Science, 598, 1:4000 for WB), rabbit anti-RFP (MBL Life Science, PM005, 1:3000 for WB). 【0125】The secondary antibodies are as follows: Alexa Fluor® labeled highly cross-adsorbed goat anti-mouse or anti-rabbit IgG antibodies (Invitrogen, #A11029, #A11031, #A11034 or #A11011, 1:500 for IF), Alexa Fluor 488 Phalloidin® (Invitrogen, #A12379, 1:1000, for IF), Alexa Fluor 647 Phalloidin (Invitrogen, #A22287, 1:500 for IF), alkaline phosphatase (AP) labeled anti-mouse IgG (Promega, #S3721, 1:10000 for WB; Sigma, #A3562, 1:30000 for WB) or AP labeled anti-rabbit IgG (Promega, #S3731, 1:10000 for WB), horseradish peroxidase (HRP) labeled anti-mouse IgG (Promega, # W4021, 1:30000 for WB), or HRP labeled anti-rabbit IgG (Promega, # W4011, 1:30000 for WB). 【0126】 Western blotting cell lysates or proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, #IPVH00010) at 24V for 1 hour using Trans-Blot® SD Semi-Dry Transfer Cell (BIO-RAD, #1703940). The blotted membranes were blocked at room temperature for 1 hour in PBS-T containing 0.05% (vol / vol) Tween®-20 and 5% (wt / vol) skim milk (PBS-T). Subsequently, the membranes were incubated overnight at 4°C in PBS-T containing 0.01% (wt / vol) NaN3 with the primary antibody described above. After washing three times with PBS-T, the membranes were incubated with AP-labeled anti-mouse or anti-rabbit IgG antibody for 1 hour. After three washes, an AP substrate consisting of 5-bromo-3-chloroindolyl phosphate (Roche Diagnostics, #10760978103) and 4-nitrobluetetrazolium chloride (Roche Diagnostics, #11383213001) was added to AP buffer and detected. 【0127】 To detect Halo7-Rac1 in EV-treated PANC-1 cells, blotted PVDF membranes were blocked overnight at 4°C with 5% (wt / vol) ECL® PRIME blocking reagent (Cytiva, # RPN418) in PBS-T, and incubated overnight at 4°C with antibodies against Rac1, Halo, or β-actin in PBS-T containing 0.5% (wt / vol) ECL® PRIME blocking reagent. After three washes with PBS-T, the membranes were incubated overnight with horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody in PBS-T containing 0.5% (wt / vol) ECL® PRIME blocking reagent. After five washes, ECL Prime Western Blotting Detection Reagent was added for detection using ImageQuant® LAS 4000 (GE Healthcare). To detect MIM in the EV fraction from FBS, PVDF membranes were incubated overnight at 4°C with an anti-MIM antibody in Can Get Signal® Solution 1 (Toyobo, #NKB-201), and then incubated overnight at 4°C with an HRP-labeled anti-mouse IgG antibody in Can Get Signal Solution 2 (Toyobo, #NKB-301). 【0128】Preparation of l-EV and s-EV fractions: EV purification was performed as previously described (T. Nishimura et al., Dev Cell 56, 842-859 e848 (2021)), with some modifications. Briefly, 30 ml of HEK293 cell suspension culture in a 50 ml centrifuge tube (Violamo, #VIO-50BN) was sequentially centrifuged at 1,000 g for 10 minutes and 3,000 g for 10 minutes at 4°C using an MX-307 centrifuge and an AR510-04 rotor (Tommy Seikou, k factor: 880) to remove cell bodies and dead cells, respectively. Next, the l-EV in the supernatant was pelletized by centrifugation at 10,000 g for 30 minutes at 4°C. The pelletized l-EV was resuspended in 1 ml of filtered PBS and centrifuged again at 10,000 g at 4°C for 30 minutes using an MX-307 centrifuge and an AR015-24 rotor (Tommy Seiki, k factor: 643). The resulting l-EV pellet was resuspended in 50 μl of PBS. The supernatant from 10,000 g, placed in a polycarbonate bottle (Beckman, #355622), was ultracentrifuged at 120,000 g at 4°C for 70 minutes using an Optima® XE-90 ultracentrifuge and a Type-45Ti rotor (Beckman, k factor: 259). This pellet was resuspended in 0.8 ml of PBS and pelletized again in an open-top thick-walled polycarbonate tube (Beckman, #343775) at 120,000 g, 4°C, for 70 minutes using an Optima TLX ultracentrifuge with a TypeTLX120.2 rotor (Beckman, k-factor: 34.8). The pelletized s-EV was resuspended in 100 μl of filtered PBS. EV preparation from FBS (30 ml) was carried out similarly, except that the FBS was heated at 56°C for 30 minutes before centrifugation to inactivate complement. 【0129】To inhibit Rac1 in extracellular viable cells (EVs), EHT1864 (TargetMol, #T6483) was added to the l-EV fraction from FBS at a final concentration of 20 μM, and the solution was incubated at 37°C for 45 minutes. After treatment, the l-EVs were pelletized by centrifugation at 10,000 g for 30 minutes at 4°C, washed with 1 ml of PBS, centrifuged again at 10,000 g for 30 minutes at 4°C, and resuspended in PBS. 【0130】 To label Halo7-tagged proteins expressed in extracellular proteins (EVs), HaloTag ligands SaraFluor® 650T (SF650T) or SaraFluor 650B (SF650B) ligands (Goryochemical, #A308-01 or #A201-01) were used. Purified EVs were incubated with 50 nM HaloTag ligand for 1 hour and shaken at 37°C and 200 rpm using an incubator-equipped shaker (BIOSAN, #TS-100C). To remove ligand from solution, EVs were washed with 1 ml of PBS, centrifuged at 10,000 g for 30 minutes at 4°C, and resuspended in PBS. For super-resolution microscopy, labeled EVs were loaded onto Amicon® Ultra 10k (Millipore, #UFC501008) and washed with 0.5 ml of PBS. 【0131】 To label extracellular fluorescein (EVs) using the PKH26 Red Fluorescent Cell Linker Kit (Sigma, #MINI26), 40 μl of the EV fraction was added to 110 μl of dilution C as instructed in the kit. Then, 150 μl of 2 μM PKH26 dye in dilution C was added and incubated at room temperature for 5 minutes. To stop the reaction, 1% (wt / vol) BSA in PBS was added and incubated at room temperature for 3 minutes. Next, 700 μl of PBS was added and the EVs were centrifuged at 10,000 g at 4°C for 30 minutes. The pelletized EVs were washed with 1 ml of PBS, centrifuged at 10,000 g at 4°C for 30 minutes, and resuspended in 40 μl of PBS. 【0132】The protein concentration of each extracellular matrix (EV) fraction was quantified using a Qubit 4 fluorometer (Invitrogen, # Q33238). The particle size distribution and particle count of each EV fraction were determined by nanotracking analysis (NTA) using a NanoSight LM10 instrument (Malvern Panalytical). Each sample was recorded five times for one minute, with the camera level set to 11 and the detection threshold set to 14. Particle concentration and standard error for each particle size were calculated using NTA3.1 software. 【0133】 EV-removed FBS was prepared by centrifuging FBS at 120,000 g for 16 hours and filtering the supernatant through a 0.22 μm filter (Merck, #SLGPR33RB). 【0134】 The effect of EV on wound healing assays was investigated using EV-deleted DMEM, including 10% EV-deleted FBS. 【0135】 PANC-1 cells and CIP4 KO PANC-1 cells were placed in DMSM containing 10% normal FBS in 2 × 10⁶ glass base dishes (IWAKI, #3910-035). 6 Cells were seeded at a number of individual cells. After 2 days of culture (the cell count approximately doubled), the cell monolayer was scratched with the tip of a plastic micropipette. After scratching, the cells were allowed to stand for 10 minutes, and images were taken at 5-minute intervals for 12 hours using CytoSMART® (Axion BioSystems). 【0136】 l-EV fraction or s-EV fraction (6 × 10) prepared from FBS 8 The particles were added after scratching to a confluent monolayer of PANC-1 cells in 1 ml of DMEM medium containing 10% (vol / vol) EV-deleted FBS in a 35 mm dish (Corning, #430165). 5 μg of total protein (approximately 1.4 × 10⁶) prepared from GFP- or GFP-MIM I-BAR expressing HEK293 cells as described above was added. 9 The l-EV fraction, containing particles or 5 μg of purified Rac1 protein, was added to the culture medium of PANC-1 cells after scratching. 【0137】To inhibit dynamin, cells were treated with either 40 μM Dynasore (CAS: 304448-55-3, AdipoGen, #AG-CR1-0045-M005) or 0.1% DMSO and incubated for 30 minutes before EV addition and cell scratching. + -To inhibit ATPase, 100 μM bafilomycin (AdipoGen, #BVT-0252-C100) or 0.1% DMSO was added to 1 ml of culture medium and incubated for 30 minutes before EV addition and cell scratching. 【0138】 The wound area was calculated using ImageJ software (NIH) to determine the area of ​​the wound covered by migrating cells at each point in time, and then measured manually. 【0139】 Immunofluorescence staining of PANC-1 cells: 5 × 10 5 Cells were seeded onto coverslips (Matsunami, #C013001) in 24-well plates. After 2 days of culture (approximate cell doubling time), once a cell monolayer had formed, the medium was replaced with DMEM without FBS, and serum starvation was performed for more than 12 hours. Subsequently, the cell monolayer was scratched using a plastic micropipette tip and injected into DMEM with 10% (vol / vol) FBS or 1.6 × 10⁶ cells. 9 The cells were cultured in 1 ml of DMEM containing EV-deficient FBS with added l-EV particles. For the wound healing assay, bafilomycin or DMSO was added 30 minutes before EV addition. 【0140】 HEK293 cells co-expressing MIM and wild-type (WT) or mutant Rac1 were incubated at 37°C for 3 hours on collagen (Toyobo, TMTCC-050) coated coverslips. 【0141】These cells were fixed in DMEM containing 10% FBS or 10% EV-removed FBS with 4% (wt / vol) paraformaldehyde (PFA) for 10 minutes at room temperature. The cells were permeable for 5 minutes using 0.1% Triton® X-100 in 1% (wt / vol) BSA-containing TBS, and then blocked in 1% (wt / vol) BSA-containing TBS for 1 hour. After washing with PBS, the cells were incubated with primary antibody diluted in 0.1% (wt / vol) BSA-containing TBS at room temperature for at least 2 hours. After washing, the cells were incubated with AlexaFluor-labeled secondary antibody and Alexa488 or Alexa647-labeled phalloidin (Invitrogen, diluted 1:500) diluted in 0.1% (wt / vol) BSA-containing TBS at room temperature for 1 hour. After further washing, the cells were mounted on ProLong® Diamond using DAPI mounting solution (Invitrogen, #P36971). 【0142】 Images were acquired using a confocal microscope (Olympus, FV1000D) with 60× or 100× oil immersion objective lenses (NA = 1.35 or 1.40, respectively). The images were analyzed using ImageJ software. Line profiles of fluorescence intensity were measured using "Plot Profile" on the indicated lines. 【0143】 The fluorescence intensity of SF650T-labeled Halo7-Rac1 released from endosome EVs into the cytoplasm of recipient cells was measured as follows: Because Halo7-Rac1 remained in the endosome EVs, Halo7-Rac1 punctures were detected as particles with a threshold of 3000 or more using the "Analyze Particles" plugin in Image J. The number of detected punctures was approximately the same as the number of Halo7-Rac1 dots counted manually. The average intensity of Halo7-Rac1 in the cytoplasm, excluding the point regions of the EVs, was measured. 【0144】Fluorescence Quantification of EVs: l-EVs were labeled with PKH26 dye and SF650T halo ligand and incubated in 1.2 ml of FreeStyle® 293 Expression Medium at 37°C for 2 hours. After incubation, EVs were observed using a confocal microscope FV1000D (Olympus) equipped with a 100× oil immersion objective lens (NA=1.40). The image pixel size was 124 nm × 124 nm (0.015 μm). 2 ) 【0145】 The acquired images were analyzed using ImageJ software. EVs were detected in the PKH26 channel using the "Analyze Particles" plugin in ImageJ. The mean fluorescence intensity of the GFP and Halo (SF650T) channels was measured in the detected particle region. The fluorescence intensity distribution of EVs was analyzed using Flowjo® software (Becton Dickinson). Linear regression lines of PKH26 (x-axis) versus other fluorescent proteins (y-axis) for EVs from single-transfected cells (e.g., GFP-MIM-I-BAR from Halo7-Rac1 transfected EVs) were used to distinguish between different fluorescence, and the standard deviation (SD) of the signal from the regression line of another fluorescence channel was used as a cutoff line for positive particles of another fluorescence. From the signal distribution, the SD was 2.5×SD for GFP-MIM-I-BAR, FKBP-YFP-MIM-I-BAR, and Halo-Rac1 channels, and 1.5×SD for the Halo-Cas12f channel. 【0146】 The size (diameter) of each EV was calculated from the pixel area using the "Analyze Particles" plugin, assuming all EVs were spherical. In the PKH26 channel observed with an objective lens (NA=1.40), the image resolution was approximately 0.5 × 568 / 1.40 = 272 nm, given the wavelength of the irradiated laser was 568 nm. Measurements were taken by combining areas of 1-3 pixels. 【0147】Labeling Efficiency and Measurement of Halo-Rac1 Protein in EVs To measure the protein concentration in EVs, droplets of SF650T Halo ligand solution were prepared in a manner similar to that previously reported (C. Sun, L. Liu, HN Vasudevan, KC Chang, AR Abate, Anal Chem 93, 9974-9979 (2021)). Briefly, 1 μl of SF650T Halo ligand solution at concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mM were added to mineral oil (Sigma, M5904-5 ml) and vortexed for 10 minutes to create droplets for measurement. According to the manufacturer (Goryō Chemical), the fluorescence intensity of SF650T Halo ligand alone was 1 / 0.076 of the fluorescence intensity in the presence of HaloTag protein. 【0148】To measure labeling efficiency, the fluorescence intensity of Halo-Rac1 in cell lysates was compared to that of SF650T Halo ligand. Untransfected or Halo-Rac1-expressing HEK293 cells were centrifuged at 10,000 g at 4°C for 30 minutes. The pellet was incubated with 50 nM HaloTag ligand for 1 hour, rotating at 37°C at 200 rpm. To remove ligand from solution, the pellet was washed with PBS, centrifuged at 10,000 g at 4°C for 30 minutes, and resuspended in sonication buffer containing 50 mM HEPES (pH 7.4), 25 mM NaCl, 1 mM EDTA, 1 mM MgCl2, and 0.5% Triton X-100. This suspension was sonicated using a VP-5S (TAITEC) sonic homogenizer at level 9 (maximum level) for 10 minutes in cycles of 10 seconds of sonication followed by 10 seconds of rest. Subsequently, the lysates were centrifuged at 17900 g (15000 rpm) for 5 minutes at 4°C (Tommy Seikou, KITMAN-18). The recovered supernatant was filtered using a 100 kDa ultrafiltration membrane (Millipore, #Ultrafree-C3HK) to remove high molecular weight factors. The pass-through fraction was concentrated using Amicon Ultra 3k (Millipore, #UFC500396). Fluorescence was measured using a fluorescence spectrometer FP-6500 (JASCO). Lysates of Halo-Rac1-expressing cells were diluted with sonication buffer, and the total Halo-Rac1 concentration (including labeled and unlabeled Halo-Rac1) determined by Western blotting compared with purified Rac1 was adjusted to 0.25 μM. Lysates of non-transfected cells were similarly diluted. SF650T Halo ligand was diluted to 0.25 μM with sonication buffer. Figure 13D shows the fluorescence spectra obtained by subtracting the lysate of untransfected cells from the lysate of Halo-Rac1-expressing cells, and the fluorescence spectra obtained by subtracting the fluorescence spectrum of the sonication buffer alone from the fluorescence spectrum of the SF650T ligand. The fluorescence intensity of fully labeled Halo-Rac1 was estimated from the fluorescence intensity of the SF650T Halo ligand divided by 0.076.Since the SF650T halo ligand and the SF650B halo ligand differ only slightly in their chromophores, their labeling efficiencies are considered to be similar. Therefore, the same labeling efficiency was assumed when calculating the number of Halo-Rac1s in EV from STORM images. 【0149】 Live cell imaging for EV uptake: CIP4 KO PANC-1 cells stably expressing CIP4-mCherry are placed in glass-based dishes in 2 × 10⁶ units. 6 Cells were seeded individually. Two days after culturing (approximate cell doubling time), the cell monolayer membrane was scratched using the tip of a plastic micropipette, and a wound healing assay was performed. Subsequently, the medium was replaced with DMEM containing 10% EV-removed FBS medium, and 3.5 × 10⁶ cells were added to 200 μl. 9 We incorporated several EVs. Time-lapse images were acquired in a chamber containing 5% CO2 at 37°C using a laser confocal microscope (FV1000D; Olympus) equipped with a 100× oil immersion objective lens (NA = 1.40; Olympus). The images were analyzed using ImageJ software. 【0150】 To observe fixed cells incorporating EVs using a 3D super-resolution microscope, 1 × 10⁶ PANC-1 cells stably expressing mEos4b-Lamp1 were prepared two days before EV addition. 5 The cells were seeded in a glass dish. As described above, l-EVs co-expressing Halo7-Rac1 and FLAG-MIM I-BAR were prepared and labeled with Halo ligand SF650B. Bafilomycin or DMSO was added 30 minutes before EV addition under the same conditions as the wound healing assay, and 2 × 10⁶ cells were added. 9Labeled l-EV particles were added to PANC-1 cells in 200 μl of DMEM containing 10% EV-deficient FBS and incubated for 3 hours. Cells were fixed at room temperature for 10 minutes using 4% PFA and 0.2% glutaraldehyde in a buffer containing 30 mM HEPES (pH 7.4), 100 mM NaCl, and 2 mM CaCl2. After washing three times with PBS, cells were treated with 0.1% NaBH4 in PBS at room temperature for 7 minutes. After washing twice with PBS, cells were stored at 4°C until observation in 1% (wt / vol) polyvinyl alcohol and 10 mM cysteamine in PBS. Super-resolution microscopy observations were performed using the reported procedure (N. Olivier, D. Keller, P. Gonczy (o with umlaut), S. Manley, PLoS One 8, e69004 (2013); M. Tachikawa et al., Sci Rep 7, 7794 (2017)) with some modifications. Immediately before observation, cells were immersed in PBS supplemented with 10 mM Tris HCl (pH 7.5), 10% glucose, 10 mM cysteamine, 50 mM 2-mercaptoethanol, 2.5 mM protocatechuic acid, 2 mM cyclooctatetraene, and 50 mM protocatechuic acid dioxygenase. An N-STORM (Nikon) super-resolution microscope equipped with a 100× / NA=1.49 objective lens (Apo TIRF 100×Oil DIC N2 Nikon) and an EMCCD camera (iXon Du-897; ANDOR) was used to obtain 20,000 images at 256×256 pixels (40.96×40.96 μm) with an exposure time of 20 ms. 15,000 fluorescence images were acquired sequentially; the first 7,500 were images of mEos4b-Lamp1, and the remaining 7,500 were images of SaraFluor 650B-labeled Halo7-Rac1. A 405 nm laser was used to activate mEos4b, and 488 nm and 647 nm lasers were used to detect Lamp1-mEos4b and SaraFluor 650B, respectively. The acquired images were analyzed using NIS-Elements software (Nikon).In 20 images of PANC-1 cells in which mEos4b-Lamp1 surrounds Halo7-Rac1 expressing EVs, five EVs were randomly selected from within the cells. As shown in Figure 2(D), the number of EVs in which the Halo7-Rac1 signal crosses around mEos4b-Lamp1 was examined, and the number of signals from Halo-Rac1 clusters within endosomes is shown in Figure 11A. 【0151】 Purification of Rac1 protein: The Rac1 protein was expressed as EGFP-GST-Rac1 in HEK293 cells and purified by removing the EGFP-GST tag as previously described (WNI Wan Mohamad Noor et al., Sci Adv 9, eadf5143 (2023)). The pEGFP-C1-GST-Rac1 plasmid was transfected into HEK293 cells using PEI. The cells were cultured at 37°C in 8% CO2 for 2 days and harvested by centrifugation at 5350g for 10 minutes. The cell pellet was lysed in 1 ml of lysis buffer containing 10 mM Tris HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol (DTT). The mixture was sonicated at 4°C using a Q700 ultrasonic homogenizer (QSONICA), and then centrifuged at 17200 g for 5 minutes at 4°C. The supernatant was incubated with 100 μl of equilibrated glutathione Sepharose 4B beads (GE Healthcare, #17-0756-01) at 4°C for 1 hour. The sample was centrifuged at 500 g for 3 minutes to collect the beads, and the supernatant was removed. The beads were washed three times with washing buffer (10 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT). To cleave EGFP-GST, PreScission Protease (GE Healthcare) was added to 200 μl of wash buffer, and the sample was rotated overnight at 4°C. The supernatant was centrifuged at 500 g for 3 minutes at 4°C to obtain purified Rac1 protein. Protein concentration was measured using a Qubit 4 fluorometer. 【0152】To investigate whether the Rac1 protein is located on the outside or inside of the EV membrane, the EV fraction was trypsinized as previously described (T. Nishimura et al., Dev Cell 56, 842-859 e848 (2021)). Briefly, the EV fraction containing 0.32 μg of protein or purified Rac1 protein was treated with 4 μM trypsin in PBS (MP Biomedicals) at 37°C for 30 minutes. After treatment, the same amount of SDS-PAGE sample buffer was added, and the fraction was analyzed by Western blotting. The ratio of Rac1 amount after trypsinization to Rac1 amount without trypsinization was calculated by analyzing the bands in the Western blotting using Image J. 【0153】 Western blotting of internalized Rac1 in EV recipient cells To measure the amount of Rac1 contained in internalized EVs, EVs remaining on the cell membrane were removed from recipient cells by trypsin treatment, as previously described (B. Cardenes et al., Int J Mol Sci 22, (2021)). Briefly, PANC-1 cells were placed in a 6-well plate with 10 6 Individual seeds were seeded. After the cells adhered, they were washed three times with serum-free DMEM medium and cultured in serum-free medium for more than 12 hours. PANC-1 cells in 1 ml of medium were divided into 8 × 10⁶ cells. 8 Cells were cultured with individual extracellular viable cells (EVs) at 37°C under a humidified atmosphere of 5% CO2 for 1, 3, and 6 hours. Subsequently, cells were treated with 0.25% trypsin (MP Biomedicals) - 0.02% EDTA in PBS at room temperature for 5 minutes to remove EVs bound to the cell surface. After the reaction, the trypsin solution was collected and the cells were pelleted by centrifugation at 3,000 g for 10 minutes at 4°C, detaching them from the plate. After removing the trypsin solution, the cells on the dish were collected in 50 μl of SDS-PAGE sample buffer and combined with the cell pellet in the trypsin solution. 10 μl of these samples were analyzed by Western blotting. 【0154】ELISA method for Halo7-Rac1 in EV recipient cells. PANC-1 cells are placed in 6 x 10 cm dishes. 6 Individual seeds were seeded. After cell adhesion, the cells were washed three times with serum-free DMEM medium and cultured in serum-free medium for more than 12 hours. Next, the l-EV fraction (9 × 10¹⁶) was obtained from HEK293 cells co-expressing FLAG-MIM I-BAR and Halo7-Rac1. 9 The particles were added to cells in 6 ml of DMEM medium containing 10% EV-deficient FBS. The cells were cultured at 37°C for 1 or 3 hours under a humidified atmosphere of 5% CO2. 【0155】 To prepare the cell lysate, the culture medium containing l-EV was removed and washed three times with PBS. The cells were lysed with 500 μl of lysis buffer. The cells were collected with a cell scraper and passed through a 1 ml syringe (TERUMO, #1-4908-01) fitted with a 25 G needle (TERUMO, #61-9703-87) 10 times to disrupt the chromosomes. Next, the cells were sonicated for 1 minute using an ultrasonic homogenizer VP-5S (TAITEC) at level 6 (maximum level), with an on time of 10 seconds and an off time of 10 seconds. The lysate was then centrifuged at 4°C and 17900 g for 5 minutes, and the supernatant was collected. 【0156】The day before measurement, anti-HaloTag rabbit antibody was dispensed at 1 μg / ml in 100 μl PBS into a 96-well ELISA plate (Thermo, #3855) and incubated overnight at 37°C. The plate was washed three times with PBS and blocked with 3% (wt / vol) BSA in PBS for 1 hour at room temperature. After three washes with PBS, 100 μl of cell lysate or lysis buffer alone was added to the plate and incubated at room temperature for 2 hours. After three washes with PBS-T, 100 μl of anti-Rac1 antibody diluted with 0.3% (wt / vol) BSA / PBS-T was added to the plate and incubated at room temperature for 2 hours. After three washes with PBS-T, 100 μl of HRP-labeled anti-mouse IgG antibody (Invitrogen) diluted 1:500 in PBS-T containing 0.5% (wt / vol) ECL Prime blocking reagent was added to the plate and incubated at room temperature for 1 hour. After washing three times with PBS-T, 100 μl of TMB solution (Thermo, 1-step Slow-TMB-ELISA, #34024) was added to the plate and incubated at room temperature. The HRP reaction was stopped by adding 100 μl of 1 M phosphate, and the absorbance at a wavelength of 450 nm was measured using a microplate reader (TriStar LB942). 【0157】 l-EVs co-expressing FLAG-MIM I-BAR and Halo7-Rac1 were serially diluted with lysis buffer, and the amount of Rac1 was measured simultaneously with the cell lysates using ELISA. The same l-EV fraction was Western-blotted with purified Rac1 protein of known concentration, and the Rac1 concentration in the l-EV was calculated. A calibration curve was created by measuring l-EV fractions with known Rac1 concentrations. To calculate the Halo7-Rac1 concentration in PANC-1 cell lysates treated with the l-EV fraction, the absorbance at 450 nm of the cell lysates was subtracted from the absorbance without the l-EV fraction, and this was divided by the slope of the calibration curve. 【0158】Cas12f reporter cells were generated using a Cas12f reporter assay lentivirus system. Lentiviruses were generated by transfecting pKLV2-mCherry_TTR_GFPGFPv3 (mentioned above) with psPAX2 (Addgene, #11260) and pMD2G (Addgene, #11259). The supernatant was filtered and added to cells. Transfected cells were selected by hygromycin-activated fluorescence cell sorting. 【0159】 Cas12f reporter cells were present at a density of 2.5 × 10⁶ cells per well in a 96-well plate (TrueLine, TR5003). 4 Cells were suspended in 100 μl of serum-free DMEM medium. 3 × 10⁶ cells were suspended in 5 μl of serum-free DMEM medium. 9 EV was added to Cas12f reporter cells and incubated at 37°C for 1 hour in a humidified atmosphere of 5% CO2. After incubation, 100 μl of DMEM medium containing 10% EV-removed FBS was added and the cells were cultured for 3 days before observation. EV-treated cells were observed using an epifluorescence microscope (Ti2 (Nikon)) equipped with a 63× lens (NA=1.4, Nikon) or an IX81 (Olympus) equipped with a 10× objective lens (NA=0.25, Olympus). Images were analyzed using ImageJ software. The number of GFP-positive cells was counted in a 2625.6 μm × 2625.6 μm area (approximately 5000 cell seeding). 【0160】 To investigate the effects of heating or freezing on extracellular viable cells (EVs), the same number of EVs were aliquoted and heat-treated in a block incubator (ASTEC, BI-516S) at 37°C for 30 minutes or 56°C for 30 minutes, followed by storage at 4°C for 1 day, or freezing at -80°C for 1 day, or freezing in liquid nitrogen followed by storage at -80°C for 1 day. After these treatments, the number of EVs was measured using NTA. The EVs were added to cells at the same EV and cell concentrations as described above. 【0161】Statistical analysis: Statistical significance between two groups was measured using Student's t-test for the specified number of experiments. Statistical significance for three or more groups was performed using one-way ANOVA corrected for Tukey's HSD (honestly significant difference) test. A value of p < 0.05 was considered statistically significant. All data are described as mean ± SD. 【0162】 (Results) Example 1: The necessity of EV endocytosis in serum-induced cell migration Endocytosis is carried out by the formation and cleavage of cell membrane entry via BAR domain superfamily proteins in cooperation with dynamin (SM Ferguson, P. De Camilli, Nat Rev Mol Cell Biol 13, 75-88 (2012); HT McMahon, E. Boucrot, Nat Rev Mol Cell Biol 12, 517-533 (2011)). Cdc42-interacting protein 4 (CIP4) possesses an F-BAR domain and is involved in endocytosis (T. Itoh et al., Nat Commun 14, 4602 (2023); E. Crosas-Molist et al., Physiol Rev 102, 455-510 (2022)), as well as in the migration of several cancer cells, including pancreatic cancer cells (W. Saengsawang et al., Curr Biol 22, 494-501 (2012); CS Pichot et al., Cancer Res 70, 8347-8356 (2010); Y. Rolland et al., Dev Cell 30, 553-568 (2014); P. Truesdell et al., Oncogene 34, 3527-3535 (2015); G. Malet-Engra et al., Cancer Res 73, 3412-3424 (2013)). 【0163】In conventional cell culture methods, fetal bovine serum (FBS) is heated at 56°C for 30 minutes to inactivate the complement system, and then cryopreserved. Treated FBS is effective in promoting cell migration. Pancreatic cancer PANC-1 cells migrate in a serum-dependent manner (I. Roy et al., Cancer Res 75, 3529-3542 (2015)). To investigate the potential role of serum extracellular viable cells (EVs) in the migration of PANC-1 wild-type (WT) and CIP4 knockout (KO) cells, EVs were depleted from thermoactivated serum by ultracentrifugation (Figure 1A, Figure 5). In wound healing assays, CIP4 knockout cells showed slower migration than WT cells in a medium containing the original serum with EVs (Figure 1B, Figure 6A). Interestingly, in a medium from which EVs had been removed, the migration of WT cells decreased to the same level as that of CIP4 KO cells (Figure 1B). Therefore, when cells possess CIP4, serum EVs contribute to cell migration. 【0164】To clarify the role of serum extracellular viable cells (EVs) in cell migration, EVs prepared from fat-burned steroids (FBS) were added to a wound healing assay. EV fractions were prepared by stepwise centrifugation and separated into large EV fractions (l-EVs) and small EV fractions (s-EVs) according to their size (C. Thery et al., J Extracell Vesicles 7, 1535750 (2018)). Particle tracking analysis of the EV size distribution showed that the diameter of the major population of serum EVs in the l-EV fraction was approximately 140 nm, while that of the serum s-EV fraction was approximately 100 nm (Figure 6B). The number of particles in the l-EV fraction derived from FBS was lower than that in the s-EV fraction (Figure 6C). These serum EV fractions were added to a wound healing assay under FBS with EVs removed. The serum l-EV fraction promoted migration of WT cells compared to CIP4 KO cells (Figure 1C). In contrast, the migration of WT cells and CIP4 KO cells under serum s-EV fraction conditions was similar (Figure 6D). To further investigate whether the effect of EVs is endocytosis-dependent, cells were pretreated with the dynamin inhibitor Dynasore. Dynasore reduced the migration of WT cells to the serum l-EV fraction, but did not reduce the migration of CIP4 KO cells (Figure 1C). These results suggest that serum l-EVs are endocytized to induce cell migration. 【0165】The inventors previously reported that l-EVs from HEK293 cells contain Rac1 and promote recipient cell migration in a MIM-dependent manner (T. Nishimura et al., Dev Cell 56, 842-859 e848 (2021)). Therefore, l-EV fractions were prepared from MIM I-BAR domain-expressing HEK293 cells (T. Nishimura et al., Dev Cell 56, 842-859 e848 (2021)), and these EVs were applied to WT cells and CIP4 KO cells in the same manner as described above (Figure 1D). Migration of WT cells under the l-EV fraction from MIM I-BAR-expressing cells was faster than that of CIP4 KO cells (Figure 1E). Furthermore, dinosaur treatment reduced the migration of WT cells under these EVs without affecting the migration of CIP4 KO cells (Figure 1E). Therefore, MIM-I-BAR-dependent l-EVs induced cell migration via endocytosis, similar to serum l-EVs. 【0166】 To elucidate the role of Rac1 in these EVs, the amount of Rac1 in serum EV fractions was compared with that in HEK293 cell-derived EV fractions. Rac1 was found in l-EVs derived from FBS, as well as in l-EVs derived from HEK293 cells expressing the MIM I-BAR domain, but not in s-EVs derived from FBS (Figure 1F, Figure 6E). Furthermore, since l-EVs derived from FBS contained MIM (Figure 1G), it is highly likely that l-EVs derived from MIM in serum also contain Rac1. 【0167】To investigate whether Rac1 in extracellular vesicles (EVs) is involved in cell migration, its activity was modified using Rac inhibitors or Rac1 mutants. First, l-EV fractions from FBS were treated with the Rac inhibitor EHT1864. After washing off EHT1864, cell migration under the inhibitor-treated EVs was examined. Interestingly, unlike untreated EVs, EHT1864-treated EVs from FBS did not affect the migration of WT cells and CIP4 KO cells (Figure 1H). Next, Rac1 mutants were encapsulated in l-EVs by co-expressing constitutively active Rac1 G12V or dominant-negative Rac1 T17N with the MIM I-BAR domain in HEK293 cells. These Rac1 mutants localized to MIM I-BAR-induced filopodia (Figure 7A) and did not affect the size distribution, number of EVs, or number of Rac1s in l-EVs (Figures 7B-7D). On the other hand, migration of WT cells under l-EVs containing Rac1 T17N was slower than migration under EVs containing Rac1 or Rac1 G12V (Figure 7E). Furthermore, the addition of purified Rac1 protein alone did not induce faster cell migration of WT cells compared to CIP4 KO cells (Figure 7F). These results suggest that Rac1 protein encapsulated in serum l-EVs or HEK293-derived l-EVs promotes cell migration. 【0168】 Example 2: To understand the mechanism of action of Rac1 in EVs in recipient cells, including internalization, endosomal transport, and release into the cytoplasm, the localization of Rac1 in EVs during intracellular transport from endocytosis to endosomes was visualized. GFP-MIM-I-BAR l-EVs added to PANC-1 cells expressing mCherry-CIP4 were observed using a confocal microscope. EVs monitored by GFP-MIM I-BAR transiently co-localized with mCherry-CIP4 on the cell membrane and gradually disappeared, suggesting endocytosis of EVs (Figure 8). 【0169】Endocytotic vesicles are encapsulated from the cell membrane and then transported to endosomes. Next, the localization of endogenous EVs containing Halo-Rac1 and mCherry-MIM I-BAR was examined at various times after EV incubation (Figure 9A). These EVs were taken up into WT recipient cells within 3 hours, while uptake was impaired in CIP4 KO cells (Figures 9B–9D). Live-cell imaging showed that EVs visualized with Halo-Rac1 and mCherry-MIM co-localized with EEA1 and Lamp1, markers for early and late endosomes, respectively (Figures 2A, 9E, and 9F). Co-localization of Halo-Rac1 with EEA1 or Lamp1 in EVs increased with time after EV addition in WT cells, but this increase was suppressed in CIP4 KO cells (Figures 9G and 9H). Importantly, in WT cells, Halo-Rac1 colocalized with EEA1 earlier than Lamp1 (Figures 2A and 2B). The distribution of internalized EVs gradually changed over time from near the cell periphery to the central nucleus, which correlated with the distribution of early and late endosomes, respectively (Figure 9I). Overall, EVs containing Rac1 were first transported to early endosomes and then to late endosomes in recipient cells after endocytosis. 【0170】Colocalization of Rac1 and Lamp1 was revealed 3 hours after EV addition, and cell migration induced by EV was observed 3 hours after EV addition (Figure 9J). Therefore, late endosomes were suggested to be a site where Rac1 escapes into the cytoplasm to promote cell migration. The localization of Rac1 from EVs in late endosomes was investigated using super-resolution microscopy with mEos4b-Lamp1-expressing recipient cells and Halo-Rac1-labeled EVs. Part of the EV-derived Halo-Rac1 signal crossed over with the Halo-Rac1 signal in Lamp1-positive late endosomes, suggesting that Rac1 in EVs was released into the cytoplasm (Figure 2C, arrowhead, and Figure 2D). In fact, EV-derived Halo-Rac1 was detected at the leading edge of migrating cells during wound healing (Figure 10A), and this was confirmed by the localization of the lamellipodia-forming protein WAVE2 (Figure 10B). Therefore, EV-derived Rac1 was considered to function in cell migration in the lamellipodia. 【0171】 To suppress endosomal function, H increases the pH of endosomes. + Cells were treated with bafilomycin, an ATPase inhibitor. Bafilomycin treatment was observed to reduce Rac1 crossover (Figure 2C, Figure 2D) and increase the number of Halo-Rac1 signals per EV retained in endosomes (Figure 11A). Furthermore, the amount of Halo-Rac1 as measured by cytoplasmic fluorescence intensity and cell migration were observed to decrease in CIP4 KO cells and after bafilomycin treatment (Figures 11B-11F). These results suggest that Rac1 in endocytized EVs is released from late endosomes, inducing cell migration. 【0172】Example 3: Stoichiometric Characteristics of Exogenous Rac1 in EVs Inducing Cell Migration The increase in cell migration by l-EVs derived from FBS was similar to that by EVs derived from the culture medium of HEK293 cells. Notably, the increase in cell migration by these EVs was at the level of native FBS containing EVs, indicating that EVs are involved in the increase in cell migration (Figure 12A). Therefore, it is essential to determine the amount of Rac1 from EVs delivered to recipient cells. EVs expressing Halo-Rac1 and MIM I-BAR were added to WT or CIP4 KO PANC-1 cells. After mild trypsin treatment to remove EVs that bound to the surface without internalization, recipient cells were harvested and the amount of internalized Halo-Rac1 was measured by Western blotting. The amount of internalized Halo-Rac1 increased dramatically in a time-dependent manner in WT cells but did not increase in CIP4 KO cells (Figure 3A). 【0173】 Using purified Rac1 protein as a reference (Figure 12B), the number of Halo-Rac1 molecules delivered via EVs (Figure 3B) and the number of endogenous Rac1 molecules in recipient cells were determined by Western blotting (Figure 12C). After 3 hours of EV incubation, the number of Halo-Rac1 molecules from EVs in WT recipient cells was 6.2 ± 0.8 × 10⁶ per cell. 4 While the amount was 1 / 10 (Figure 3B), the amount of endogenous Rac1 molecules per cell was 1.2 ± 0.3 × 10⁶ 7 The number of cells was 0.5 ± 3.0 × 10¹⁴ (Figure 12C). Therefore, the ratio of incorporated Halo-Rac1 to endogenous Rac1 in cells after 3 hours was 0.53 ± 0.14% (Figure 3C; summarized in Figure 3D). Sandwich ELISA using anti-HaloTag antibody and anti-Rac1 antibody was performed using purified Halo-Rac1 protein as the standard (Figure 12D, E). After 3 hours of EV incubation, the number of Halo-Rac1 molecules incorporated into recipient cells was 5.5 ± 3.0 × 10¹⁴ by ELISA. 4This was confirmed by measurements obtained using Western blotting (Figure 12F). Untagged Rac1 from donor cells was also packaged in EVs in amounts comparable to Halo-Rac1 (Figure 3A), at 4.8 ± 3.6 × 10⁶ per cell. 4 It was estimated that (Figure 3D). The total amount of exogenous Rac1 delivered through these EVs was 1.1 ± 0.9 × 10¹⁶ per cell. 5 (Figure 3D), and the ratio of incorporated Rac1 (Halo-Rac1 from EVs and untagged Rac1) to intracellular Rac1 was approximately 1% (Figure 3D). 【0174】 Next, as summarized in Figures 3D and 12G, the number of Rac1 molecules in EVs from FBS and in EVs from the culture medium of HEK293 cells expressing Halo-Rac1 and MIM was measured. The EV concentrations were similar between FBS and HEK293 cell culture medium (Figures 6C and 7C). The average number of Rac1 molecules per EV was also similar, with 5.2 ± 1.4 × 10¹⁶ molecules in l-EVs derived from FBS. 2 Molecular weight (Figure 6E), l-EV derived from HEK293 medium: 4.5 ± 1.7 × 10⁻⁶ 3 The molecule was (Figure 7D). Of the Rac1 molecules in EVs from HEK293 cells, Halo-Rac1 was present in 2.5 ± 1.7 × 10¹⁶ molecules per EV. 3 It was a molecule. Western blotting revealed that the amount of internalized Halo-Rac1 molecules per cell was 6.2 ± 0.8 × 10⁻⁶. 4 Since it was a molecule (Figure 3B), it was estimated that 25 ± 16 EVs, representing 3.1 ± 2.1% of the added EVs, were internalized. 【0175】 Next, the population of EVs possessing Halo-Rac1 was measured under microscopic observation (Figure 13A). Halo-Rac1-positive EVs were defined as those with a Halo signal higher than the standard deviation of 2.5 for fluorescence of EVs in cells without Halo-Rac1 (cutoff line in Figure 13B). EVs expressing both MIM and Halo-Rac1 were estimated to account for approximately 9% of all EVs observable under fluorescence microscopy (Figure 13B). The average number of Halo-Rac1 cells per EV, determined by Western blotting, was approximately 2.5 × 10⁶. 3It was a molecule (summarized in FIGS. 7D and 12G). Considering the above expression rate, EV was about 2.5×10 3 / 0.09 = 2.8×10 4 It was estimated to have a Halo-Rac1 molecule (FIG. 13G). 【0176】 Next, the number of Halo-Rac1 molecules in each EV was directly determined by fluorescence intensity. Droplets of the SF650T haloligand solution were used as a standard, and in this solution, fluorescence increased linearly depending on the haloligand concentration (FIG. 13C). As a result of comparing the fluorescence intensity of the SF650T haloligand with the fluorescence intensity of Halo-Rac1 in cells treated with SF650T, the labeling efficiency of Halo-Rac1 was 24.4±5.3% under the conditions of the present inventors (FIG. 13D). Using the correlation between the fluorescence intensity of Halo-Rac1 and the Halo-Rac1 concentration (FIG. 13E, left), and the relationship between the EV size of Halo-Rac1 / GFP-MIM double-positive EVs (FIG. 13E, right), when calculating the average number of SF650T-labeled Halo-Rac1 per EV, it was about 5.5×10 3 molecules, and with the above labeling efficiency, the Halo-Rac1 concentration per EV was about 2.3×10 4 molecules (FIGS. 13F and 13G). 【0177】 With a super-resolution microscope, Halo-Rac1 in EVs in recipient cells was observed under on-off dye SF650B haloligand labeling. As a result, in the endosomes of cells treated with bafilomycin, 239±144 signals of SF650B were observed, and the cytoplasmic release of Halo-Rac1 from EVs was inhibited (FIG. 11A). Since the SF650B molecule emits light about 20 to 100 times in 450 seconds (S. N. Uno et al., Nat Chem 6, 681-689 (2014)), it is considered that SF650B-Halo-Rac1 emits 6.7 to 33 signals during 150 seconds of observation. Assuming that the labeling efficiency of SF650B is the same as that of SF650T, 1.5±0.9×10 2This means that (=240 / 0.244 / 6.7) or fewer Halo-Rac1 molecules will be present in EVs captured by endosomes. However, Halo ligands are known to be difficult to detect due to their high density, and the detection of signals may be significantly reduced (H. Takakura et al., Nat Biotechnol 35, 773-780 (2017)). Alternatively, the size of the endosomal cup is approximately 200 nm (Y. Yu, SH Yoshimura, Nat Commun 14, 4602 (2023)), which corresponds to a common size of EVs of approximately 200 nm, so smaller EVs may be preferentially endocytized (Figures 6B and 7B). 【0178】 Example 4: Engineering of Cell Membrane-Derived EVs To demonstrate direct protein transport by MIM-dependent EVs, the genome editing enzyme Cas12f (T. Hino et al., Cell 186, 4920-4935.e4923 (2023)) was packaged into MIM-dependent EVs by rapamycin-induced binding of FKBP and FRB (T. Komatsu et al., Nat Methods 7, 206-208 (2010)) (Figure 4A). After co-expression of MIM I-BAR-FKBP, Cas12f-Halo-FRB, and guide RNA in HEK293 cells, rapamycin treatment did not change the number of EVs, but increased the amount of Cas12f in the l-EV fraction (Figure 4B). The number of Cas12f molecules per EV was determined by Western blotting (Figure 14B). The average number of Cas12f molecules per EV determined by Western blotting was 0.65 ± 0.16 before rapamycin treatment and 1.53 ± 0.38 after rapamycin treatment (Figure 14C). Therefore, chemically induced dimerization increased the number of Cas12f molecules present in the l-EV fraction by 2.4 times. 【0179】Next, to detect errors prone to Cas12f-mediated genome editing, reporter cells with a frameshifted GFP coding sequence were prepared, in which the Cas12f target sequence between mCherry and GFP was cleaved, and then GFP was translated by the addition or deletion of nucleotides (Figure 4A). Reporter cells prepared from HEK293 or PANC-1 cells were treated with EVs containing Cas12f, and fluorescence was observed. The number of GFP-positive reporter cells after the addition of the l-EV fraction increased proportionally to the amount of Cas12f delivered (Figures 4C, 4D). EVs from rapamycin-treated cells were added at a rate of 1.2 × 10⁶ per recipient cell. 5 When added to recipient cells via EV, 0.77±0.16% of HEK293 cells and 0.49±0.10% of both HEK293 and PANC-1 cells became GFP-positive (Figures 4C and 4D). 【0180】 Similarly, after adding Cas12f-carrying EVs to reporter cells (Figure 15A) and gingival cancer Ca9-22 cells (Figure 15B) generated from HeLa cells, GFP-positive cells were observed, suggesting the robustness of EVs in delivering Cas12f. However, the generation of GFP-positive cells by EVs was infrequent in these reporter cells and was highest in HEK293 cells, suggesting a cell type preference for EV incorporation. 【0181】We investigated whether the delivery of Cas12f via EVs was MIM-dependent. When mutations in five lysine residues in the MIM I-BAR domain attenuated protrusion formation (S. Suetsugu et al., J Biol Chem 281, 35347-35358 (2006)), mutants of these lysine residues to alanine (5KA) did not promote EV generation (T. Nishimura et al., Dev Cell 56, 842-859 e848 (2021)). The l-EV fraction from cells expressing the MIM 5KA mutant together with Cas12f did not turn on GFP in recipient reporter cells (Figure 4D), indicating that Cas12f was delivered via MIM-dependent protrusion-derived EVs. Furthermore, CIP4 KO PANC-1 cells containing the reporter sequence did not efficiently convert to GFP-positive status after EV treatment compared to WT cells (Figure 4D and Figure 15C), indicating that MIM-induced EVs containing Cas12f were delivered in a CIP4-dependent manner, similar to those containing Rac1. 【0182】 Example 5: Stability of cell membrane-derived EVs. Thermal inactivation and Rac1 in the l-EV fraction from frozen FBS promoted cell migration (Figure 1). Next, we investigated whether Cas12f in the EV fraction retained its activity after heating and freezing, similar to thermal inactivation of FBS. EV fractions maintained at 4°C or 37°C for 30 minutes retained their ability to produce GFP-positive cells (Figures 4E and 4F). Surprisingly, EV fractions left at 56°C for 30 minutes also retained their ability to produce GFP-positive cells (Figures 4E and 4F). Furthermore, EV fractions stored at -80°C for 30 minutes, and EV fractions snap-frozen in liquid nitrogen before being placed at -80°C, also retained their ability. These results suggest that MIM-dependent and protrusion-derived EVs retain inclusion enzymes, including Rac1 and Cas12f, intact for action in recipient cells even after heating or freezing. 【0183】Example 6: Comparison of protrusion-derived EVs and endosome-derived EVs. Most EV engineering strategies for protein delivery have utilized endosome-derived EVs within the s-EV fraction (Lu Y, et al., Molecular Therapy Nucleic Acids 34, (2023); Whitley JA, et al., J Extracell Vesicles 12, e12343 (2023)). The tetraspanin protein CD63 functions as a marker for endosomes and endosome-derived EVs (Welsh JA, et al., J Extracell Vesicles 13, e12404 (2024)). To compare the efficiency of protrusion-derived EVs and endosome-derived EVs in the delivery of genome editing enzymes, FKBP was fused to either the MIM I-BAR domain or CD63 to facilitate the packaging of FRB-tagged Cas12f. From HEK293 cells transfected with these constructs, the l-EV and s-EV fractions were recovered by centrifugation under conditions optimized for the separation of each fraction. 【0184】 The Cas12f loading efficiency per EV was determined by Western blotting (Figure 16A, B). The mean number of Cas12f molecules per l-EV from MIM I-BAR-expressing cells was 1.2±0.45 and 4.3±1.8 with and without 100 nM rapamycin treatment, respectively. When Cas12f was expressed in the MIM I-BAR domain following the T2A sequence, the number of Cas12f molecules per l-EV increased to 11.2±6.9 ​​with rapamycin treatment. In contrast, CD63-mediated packaging resulted in a higher Cas12f load per EV than MIM I-BAR due to rapamycin dimerization, with mean Cas12f molecules per l-EV being 7.7±0.65 and 55.3±1.3 with and without 100 nM rapamycin treatment, respectively (Figure 16A, B). However, in s-EVs obtained from both MIM I-BAR-expressing cells and CD63-expressing cells, the Cas12f content per s-EV was lower than in l-EVs treated with rapamycin (Figure 16A, B). 【0185】 To evaluate the genome editing efficiency of Cas12f delivered by EVs, equal numbers of these l-EVs and s-EVs were applied to reporter cells. l-EVs from MIM I-BAR-expressing cells showed higher genome editing activity than those from CD63-expressing cells and the s-EV fractions from both cells (Figure 16C). It is noteworthy that the genome editing efficiency of Cas12f and CD63-expressing cell-derived EVs did not change regardless of rapamycin treatment, indicating that Cas12f actively loaded into EVs via CD63 did not function in the presence of rapamycin (Figure 16D). The genome editing efficiency per Cas12f protein showed that Cas12f packaged via MIM I-BAR retained approximately 30-fold higher genome editing efficiency than that packaged via CD63 (Figure 16D). These results indicate that cell membrane protrusions induced by MIM are better suited to packaging enzymatically active proteins into EVs compared to CD63-mediated endosomal packaging. 【0186】 (Discussion) The examples demonstrated that serum EVs contain the low molecular weight GTPase Rac1 and promote cell migration after endocytosis. The number of molecules delivered was shown to be sufficient to explain the effect of EVs on recipient cells. Furthermore, these EVs are stable to heat and freeze and can be designed as stable carriers for bioactive proteins, including genome editing enzymes. 【0187】Sufficient cytoplasmic release of EV cargo for recipient cell control. For EV cargo to function, there are two points of action: binding of EV to recipient cells and release of cargo to recipient cells. Mechanisms for cargo release from extracellular vehicles (EVs) include fusion of EVs with the cell membrane (I. Parolini et al., J Biol Chem 284, 34211-34222 (2009)) or endocytosis followed by escape to the cytoplasm (LA Mulcahy, RC Pink, DR Carter, J Extracell Vesicles 3, (2014)). Endocytosis can be clathrin-dependent endocytosis (T. Tian et al., J Biol Chem 289, 22258-22267 (2014)), phagocytosis (D. Feng et al., Traffic 11, 675-687 (2010)), or macropinocytosis (D. Fitzner et al., J Cell Sci 124, 447-458 (2011)). The MIM-dependent EVs in the examples were thought to be taken up into cells via clathrin-mediated endocytosis, given the involvement of CIP4 and dynamin. The diameters of the observed clathrin-coated pits ranged from 10 nm to approximately 300 nm on electron microscopy (I. Canton, G. Battaglia, Chem Soc Rev 41, 2718-2739 (2012)) and from 120 nm to 200 nm on atomic force microscopy (Y. Yu, SH Yoshimura, Nat Commun 14, 4602 (2023)). The sizes of l-EVs derived from FBS (Figure 6B) and l-EVs derived from MIM-expressing cells (T. Nishimura et al., Dev Cell 56, 842-859 e848 (2021)) (Figure 7B) mainly fell within the diameter range of clathrin-mediated endocytosis. 【0188】Although several reports have described the release of EV cargo proteins from endosomes after endocytosis, the functionality of the released cargo molecules was unclear. Importantly, cargo release and cell migration by Rac1-containing EVs were inhibited by bafilomycin, which inhibits endosomal function by blocking the ATP pump (Figure 2C, Figure 11F) (E. Bonsergent et al., Nat Commun 12, 1864 (2021); B. S. Joshi, M. A. de Beer, B. N. G. Giepmans, I. S. Zuhorn, ACS Nano 14, 4444-4455 (2020)). These results suggest that the endosomal release of EVs is a controlled and intact intracellular process rather than endosomal disruption. The fusion of intraluminal vesicles (ILVs) with the endosomal membrane is called reverse fusion, and its mechanism is beginning to be elucidated (E. R. Eden, C. E. Futter, Curr Biol 31, R1037-r1040 (2021); P. Perrin et al., Curr Biol 31, 3884-3893.e3884 (2021)). How the reverse fusion of ILVs of endocytosed EVs is controlled is important for future research. 【0189】 The number of Rac1 molecules delivered to the cytoplasm was thought to be sufficient. In vitro, the concentration of Rac1 required to induce actin polymerization is 0.3 - 10 μM (B. Chen et al., Elife 6, e29795 (2017); S. Suetsugu et al., J Cell Biol 173, 571-585 (2006).), and the K d for the binding of Rac1 to the WAVE regulatory complex is approximately 0.2 μM (B. Chen et al., Elife 6, e29795 (2017)). The internalized Rac1 derived from EVs (1.1 × 10 5 molecules per recipient cell, Figure 3D) was approximately 10000 μm 3 (1 × 10 -11Assuming a uniform distribution throughout the cell volume of L) (N. Walter, A. Micoulet, T. Seufferlein, JP Spatz, Biointerphases 6, 117 (2011)), the concentration would be approximately 0.02 μM. Considering a region within 2 μm of the leading edge with an arc length of approximately 50 μm (P. Lappalainen, T. Kotila, A. Jegou, G. Romet-Lemonne, Nat Rev Mol Cell Biol 23, 836-852 (2022)), the volume of the lamellipodium would be 94 μm². 3 (0.94 × 10 -13 It is estimated that L) is the case. Assuming that exogenous Rac1 is transferred to this lamellipodium, the amount of EV-derived Rac1 would be approximately 1.9 μM, assuming 100% escape from the endosome. Therefore, sufficient exogenous Rac1 molecules to activate actin polymerization by WAVE2 can be present at the leading edge even if there is little escape of EV contents from the endosome. 【0190】 If the internalization rate of Cas12f-containing EVs is similar to that of Rac1-containing EVs (3.1 ± 2.1%, Figure 3D), then the Cas12f-positive EVs will be 3.7 ± 2.5 × 10 3 It is thought that an average number of Cas12f molecules were delivered to recipient cells (Figure 14C). Considering that 1.53 ± 0.38 Cas12f molecules were produced per EV in the presence of rapamycin (Figure 14C), the average was 5.6 ± 4.1 × 10⁶. 3 This means that 100 Cas12f molecules were delivered to the recipient cells. However, after adding Cas12f-containing EVs to wild-type PANC-1 cells, 0.49% of the cells were GFP-positive (Figure 4D). If the number of EVs in the recipient cells follows a Gaussian distribution, the top 0.49% of cells would be 1.0 ± 0.02 × 10⁶. 4 It is thought that more than 10 EVs were received, and the number of Cas12f delivered from these EVs was 1.6 ± 0.4 × 10 per cell. 4The number of Cas12f molecules required for genome editing was suggested by SpCas9 delivery via electroporation. When cells were electroporated in a culture medium containing 1 μM purified SpCas9, genome editing was performed in 60–90% of the cells (S. Lin, BT Staahl, RK Alla, JA Doudna, Elife 3, e04766 (2014); Y. Wu et al., Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat Med 25, 776-783 (2019)). Electroporation can replace approximately 1% of the cytoplasm with the outer culture medium, as measured by a fluorescently labeled dextran with a molecular weight similar to SpCas9 (DA Zaharoff, JW Henshaw, B. Mossop, F. Yuan, Exp Biol Med (Maywood) 233, 94-105 (2008)). Cell volume approximately 10,000 μm 3 Assuming this, approximately 6 × 10 per cell 5 This results in the introduction of 100 SpCas9 molecules. This number is approximately 60 times higher than the expected delivery of Cas12f for genome editing, which may suggest that EV can deliver intact Cas12f more efficiently than electroporation. 【0191】The physiological significance of Rac1-containing protrusion-derived extracellular vehicles (EVs): This discovery may be useful in understanding serum-related EV-mediated physiological events, such as wound repair, cell migration into the vascular system, and the pathogenesis of tumor metastasis. MIM is expressed in most cells, especially nerve cells, kidney cells, immune cells, and endothelial cells (PK Mattila, M. Salminen, T. Yamashiro, P. Lappalainen, J Biol Chem 278, 8452-8459 (2003); J. Saarikangas et al., J Cell Sci 124, 1245-1255 (2011); MP Maddugoda et al., Cell Host Microbe 10, 464-474 (2011); N. Kim et al., Elife 13, e96891 (2024); Y. Senju et al., J Biol Chem 299, 104571 (2023)), and these are thought to be a natural source of extracellular viable cells (EVs) including Rac1. Rac1-containing extravasation vessels (EVs) are also generated from the entry pores of cancer cells (AE Sedgwick, JW Clancy, M. Olivia Balmert, C. D'Souza-Schorey, Sci Rep 5, 14748 (2015)). Therefore, EVs derived from Rac1-containing protrusions are released into the bloodstream, taken up by serum-exposed cells, and may facilitate cell migration to generate the vascular system during development and wound healing. 【0192】Comparison of delivery between cell membrane-derived EVs and endosome-derived EVs. EVs are mainly classified into two types: endosome-derived EVs (exosomes) and cell membrane-derived EVs (ectosomes) (G. van Niel, G. D'Angelo, G. Raposo, Nat Rev Mol Cell Biol 19, 213-228 (2018); JA Welsh et al., J Extracell Vesicles 13, e12404 (2024); AC Dixson, TR Dawson, D. Di Vizio, AM Weaver, Nat Rev Mol Cell Biol 24, 454-476 (2023)). Endosome-derived extracellular vesicles (EVs) are sourced from intraluminal vesicles within multivesicular endosomes (PI Hanson, A. Cashikar, Annu Rev Cell Dev Biol 28, 337-362 (2012); MF Baietti et al., Nat Cell Biol 14, 677-685 (2012); K. Trajkovic et al., Science 319, 1244-1247 (2008)), and are secreted by the fusion of endosomes with the cell membrane (M. Colombo, G. Raposo, C. Thery, Annu Rev Cell Dev Biol 30, 255-289 (2014)). Another source of extracellular vesicles (EVs) is the cell membrane, where small buds and protrusions are excised to secrete EVs (G. D'Angelo, G. Raposo, T. Nishimura, S. Suetsugu, Nat Rev Mol Cell Biol 24, 81-82 (2023); AC Dixson, TR Dawson, D. Di Vizio, AM Weaver, Nat Rev Mol Cell Biol 24, 454-476 (2023); K. Rilla, J Extracell Vesicles 10, e12148 (2021)).It should be noted that these origins of EVs are not given much consideration in the preparation of EV fractions by centrifugation and known affinity purification methods (JA Welsh et al., J Extracell Vesicles 13, e12404 (2024)). The most common experimental classes of EVs, l-EVs and s-EVs, are based on centrifugation, and there is a large overlap in their sizes (Figure 6B), although s-EVs are generally thought to be rich in endosomal EVs (JA Welsh et al., J Extracell Vesicles 13, e12404 (2024)). 【0193】 Rac1 is localized to the cell membrane via lipid modification (AJ Ridley, Curr Opin Cell Biol 36, 103-112 (2015)). The inventors found that Rac1 is present on cell membrane protrusions generated by MIM (Figure 7A). Therefore, it is thought that Rac1 is contained in the extracellular matrix (EV) by cleavage of the protrusions, and this occurs under neutral pH conditions. The expressed Rac1 interacts with the I-BAR domain of MIM by co-immunoprecipitation analysis (G. Bompard, SJ Sharp, G. Freiss, LM Machesky, J Cell Sci 118, 5393-5403 (2005); JC Dawson, S. Bruche, HJ Spence, VM Braga, LM Machesky, PLoS One 7, e31141 (2012)). Assuming that Rac1 is incorporated into EVs in endosomes, Rac1 may release GTP through protein unfolding at acidic pH (M. Koivusalo et al., J Cell Biol 188, 547-563 (2010)), suggesting that Rac1 activity may be lost in EVs. Therefore, filopodia are suitable EV sources for transporting signaling proteins like Rac1. 【0194】While not intended to be constrained by any particular theory, the neutral pH of filopodia provides an excellent environment for packaging manipulated proteins such as Cas12f. As shown in Figure 16, Cas12f packaged in endosome-derived EVs via CD63 showed higher protein loading per EV than Cas12f packaged in MIM-induced EVs, but with significantly lower genome editing efficiency. This suggests that, given that endosomes function as sites of protein degradation, CD63-mediated endosome packaging may result in protein degradation or misfolding. 【0195】 In the examples, the EV cargo proteins Rac1 and Cas12f were found to be unexpectedly stable even after heating and freezing (Figures 1 and 4). Serum for cell culture is typically cryopreserved and thermally inactivated at 56°C. The thermal denaturation temperature of Rac1 is 64.8°C (Y. Toyama, K. Kontani, T. Katada, I. Shimada, Sci Adv 5, eaax1595 (2019)). Cas12f retains dsDNA cleavage activity even above 60°C (Z. Wu et al., Nat Chem Biol 17, 1132-1138 (2021)). Therefore, the preservation of Rac1 and Cas12f in EV is due to the inherent properties of Rac1 and Cas12f, but also to the ability of EV to preserve enzymes. Packaging in EV in a neutral state within the cell membrane is undoubtedly advantageous for protein preservation. Regarding the stability of other proteins in extracellular matrix (EV) under these conditions, further investigation is needed to understand the mechanisms of protein stability during EV. 【0196】If the stability of cargo proteins can be enhanced within EVs, EVs could become an efficient delivery tool for bioactive proteins. Several studies have attempted to package genome editing enzymes into EVs, but most rely on s-EVs, which are enriched EVs derived from endosomes. In this example, packaging Cas12f via CD63 resulted in higher protein loading per EV, but genome editing efficiency was significantly lower compared to EVs mediated by MIM. 【0197】 Previous studies have reported low genome editing efficiency using s-EV-derived deliveries. For example, 1.5 × 10⁶ EVs from an s-EV fraction passively carrying Cas9 expressed in the cytoplasm were transferred to HEK293 cells. 6 When added on an individual / cell basis, genome editing occurred at 0.05% (OG de Jong et al., Nat Commun 11, 1113 (2020)). When Cas9 bound to tetraspanin via split GFP was placed in the EV of the s-EV fraction, genome editing occurred in 1 × 10 cells per cell. 5The EV rate was 0.02% (C. Zhang, R. Schekman, Elife 12, e84391 (2023)). When Cre recombinase bound to CD81 was incorporated into EVs of the s-EV fraction using the FKBP-FRB system, genomic recombination occurred only in the presence of the endosomal escape enhancer chloroquine or UNC10217938A (N. Heath et al., Nanomedicine (Lond) 14, 2799-2814 (2019)). To enhance EV delivery, EVs were modified with viral proteins. When EVs were produced in cells expressing the viral protein VSV-G, which is incorporated into EVs of the s-EV fraction, delivery became more efficient (X. Yao et al., J Extracell Vesicles 10, e12076 (2021); P. Gee et al., Nat Commun 11, 1334 (2020)). Using the FKBP-FRB dimerization system, approximately 7.9 Cas9 molecules were incorporated into EVs along with VSV-G, resulting in 1.2 × 10⁶ molecules per cell. 6 Treatment with one EV resulted in genome editing of 3% of recipient cells (P. Gee et al., Nat Commun 11, 1334 (2020)). In the example, the results were 1.2 × 10⁶ cells per cell. 5Adding 1 / 10th the amount of MIM-containing EVs and Cas12f-containing EV fractions resulted in genome editing of 0.8% of reporter HEK293 cells (Figure 4C). While there are various limitations to directly comparing these studies due to differences in DNA-cutting enzymes, the genome editing efficiency of MIM-containing and Cas12f-expressing EVs may be approximately 200 times higher than passively Cas9-supported EVs (OG de Jong et al., Nat Commun 11, 1113 (2020)) and approximately 2.5 times higher than VSV-G modified EVs actively supported by the FKBP-FRB system (P. Gee et al., Nat Commun 11, 1334 (2020)), considering the ratio of added EVs to recipient cells. Furthermore, it is important to note that while the guide RNA was passively loaded onto the EVs in the above system, actively loading the guide RNA or pre-expressing the guide RNA in recipient cells improved genome editing efficiency (P. Gee et al., Nat Commun 11, 1334 (2020); X. Osteikoetxea et al., J Extracell Vesicles 11, e12225 (2022)), suggesting that actively loading RNA is promising for the future. Overall, EVs produced by the combination of MIM and Cas12f appear to have superior delivery efficiency compared to other systems that do not involve modification by viral proteins. 【0198】 In conclusion, I-BAR protein-dependent protrusion-derived extracellular vehicles (EVs) are crucial for the transport of physiological proteins such as Rac1 for cell migration. Quantitative information on EV internalization and EV cargo release demonstrates that protrusion-derived EVs are a powerful delivery tool for any protein with engineering capabilities. 【0199】 Example 7: Investigation of cell culture conditions for EV production (Materials and Methods) Multiple cell types were cultured statically or with shaking in plates or flasks according to the following procedures 1 to 6, and the number of EVs produced under each culture condition was measured. 【0200】 1. As a model of cells and suspension cells, FreeStyle 293 cells (HEK293 adapted for suspension culture, Thermo Fisher) were cultured in serum-free FreeStyle 293 medium. Three types of adherent cells were cultured as models of adherent cells: HEK293 adherent (HEK293 in normal adherent culture), Lenti-X (trademark) (a substrain of HEK293T with high virus production, TakaraBio), and B16 melanoma cells, in 10% FCS (EV-depletion) and DMEM supplemented with penicillin and streptomycin. EV-depletion of FCS was obtained by ultracentrifugation of inactivated FCS at 120,000 g for 16 hours, and the resulting supernatant was filtered through a 0.22 μm filter to remove EVs derived from the FCS. 【0201】 2. FreeStyle 293 cells (FS293 cells), which are suspension cells, were prepared by adding 160 μL of PEI (1 μg / μL) and 20 μg of DNA to 3.3 mL of OPTI MEM, and then adding a final concentration of 2 × 10⁶ cells. 5 Cells and culture medium were added to prepare 48 mL of solution to achieve a cell / mL ratio. This mixture was then plated in a 6-well plate (2 × 10⁶). 5 cells / mL × 2 mL = 4 × 10 5 Four 12-well plates (2 x 10 cells / well) 5 cells / mL × 1 mL = 2 × 10 5 Cells were seeded in four wells, or 48 mL in a 135 mL Erlenmeyer flask, and incubated at 37°C and 8% CO2 for 72 hours under static or shaking (135 rpm) conditions. 【0202】 3. Adherent cells HEK293 adherent, Lenti-X, and B16 were seeded into four 6-well plates (0.5 × 10⁶). 5 cells / ml × 2 ml = 1 × 10 5After cell / well preparation, the cells were cultured for 24 hours at 37°C and 5% CO2, followed by transfection. For transfection, a. Mixture 1: 0.6 μg DNA + 200 μl OPTI MEM b. Mixture 2: 4 μl PEI (1 μg / μl) + 200 μl OPTI MEM c. Mixture 1 and Mixture 2 were mixed to make Mixture 3 d. Mixture 3 was added to 2 ml of culture medium e. Eight hours after transfection, the medium was replaced with 10% FCS (EV-depletion) DMEM and allowed to stand for 5 minutes. Subsequently, the cells were cultured for 72 hours at 37°C and 5% CO2 under standing or shaking (100 rpm) conditions. 【0203】 4. After culturing cells 2 or 3 for 72 hours, the culture medium or adherent cells were collected, and the number of viable cells was counted using trypan blue staining in a portion of the culture. The number of dead cells was negligible. 【0204】 5. The culture medium was subjected to stepwise centrifugation, specifically centrifugation at 1,000 g for 10 minutes, followed by centrifugation of the supernatant at 3,000 g for 10 minutes, and then centrifugation of the supernatant at 10,000 g for 30 minutes to obtain precipitate and supernatant. The 10,000 g precipitate was resuspended in PBS and centrifuged at 10,000 g for 30 minutes to obtain the large EV (l-EV) fraction. The 10,000 g supernatant was centrifuged at 120,000 g for 70 minutes, the precipitate was resuspended in PBS, and centrifugation at 120,000 g for 70 minutes to obtain the small EV (s-EV) fraction. (Reference: Hu et al., STAR Protocols 2, 100625 (2021), https: / / star-protocols.cell.com / protocols / 751.) 【0205】6. The number of EVs in each fraction was measured using the NTA particle analyzer, and the number of EVs produced per cell was determined. The relative EV counts for the l-EV fraction obtained from each culture medium are shown in Figures 17(A) and 17(B), and the relative EV counts for the s-EV fraction are shown in Figures 18(A) and 18(B). The number of EVs per cell in the l-EV fraction is shown in Figures 19(A) to 19(C), and the number of EVs per cell in the s-EV fraction is shown in Figures 20(A) and 20(B). 【0206】 As shown in Figures 17 to 20, l-EVs showed a greater increase in EV number than s-EVs under shaking culture. The rate of increase differed between flasks and plates, suggesting a possible dependence on the shape and material of the container, but l-EVs increased with shaking regardless of the cell type. 【0207】 The present invention has been described above based on embodiments. These embodiments are illustrative, and it will be understood by those skilled in the art that various modifications are possible in combinations of their components and processing processes, and that such modifications also fall within the scope of the present invention.

Claims

1. One or more combinations of a first engineered polypeptide comprising a multimerizing sequence I and a cargo entity, and a second engineered polypeptide comprising a multimerizing sequence II capable of associating with the multimerizing sequence I and an I-BAR (Inverse-Bin / Amphiphysin / Rvs) domain.

2. The combination according to claim 1, wherein the polymerization of the polymerizing sequence II and the polymerizing sequence I is induced by a low-molecular-weight compound or light.

3. The following combinations of the aforementioned multimerization sequence I and the aforementioned multimerization sequence II: (1) GAI and GID1 (gibberellin insensitive dwarf 1), (2) Abscisic acid insensitivity 1 (ABI) and pyrabactin resistance-like (PYL) protein, (3) Abscisic acid insensitivity 1 (ABI) and PYR Mandi The combination of claim 1 is one or more combinations selected from (4) FKBP and the FRB domain, (5) FKBP and Calcineurin A, (6) FKBP and CyP-Fas, (7) iLID and LOVssrA-SsrB, (8) LOVTRAP and LOV2-Zdk, and (9) the photoreceptor cryptochrome 2 (CRY2) and the CRY-interacting basic helix-loop-helix 1 (CIB1) protein.

4. The combination of claim 1, wherein the I-BAR domain is one or more I-BAR domains selected from MIM, IRSp53, IRTKS, Pinkbar, and ABBA.

5. A polynucleotide encoding a first manipulated polypeptide as described in claim 1, a polynucleotide encoding a second manipulated polypeptide as described in claim 1, and one or more combinations thereof of these polynucleotides.

6. One or more vectors comprising the combination described in claim 5.

7. A host cell comprising the vector according to claim 6.

8. A method for loading a first manipulated polypeptide according to claim 1 and a second manipulated polypeptide according to claim 1 into lipid bilayer particles, comprising culturing the host cells according to claim 7.

9. The method according to claim 8, comprising culturing the host cells described in claim 7 that have been in contact with a multimerization-inducing factor.

10. A method for producing lipid bilayer particles comprising a first manipulated polypeptide according to claim 1 and a second manipulated polypeptide according to claim 1, comprising recovering them from the culture supernatant of a host cell according to claim 7.

11. A method for producing lipid bilayer particles comprising the first manipulated polypeptide according to claim 1 and the second manipulated polypeptide according to claim 1, comprising the following steps: (1) culturing the host cells according to claim 7 in a culture medium in the presence of a polymerization-inducing factor, and (2) recovering the culture supernatant from the culture medium according to step (1).

12. An engineered lipid bilayer particle comprising a first engineered polypeptide according to claim 1 and a second engineered polypeptide according to claim 1.

13. A pharmaceutical product comprising lipid bilayer particles as described in claim 12.

Images