Engineered extracellular vesicles

Abstract

Described herein is a composition of engineered extracellular vesicles for the delivery of cargo to targeted tissues and cells.Described herein is also a method for making and using the extracellular vesicles described herein.Finally, described herein is a method for treating a subject, comprising administering to the subject the extracellular vesicles described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 183,749, filed May 4, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING The sequence listing, an 854 KB ASCII text file created on May 3, 2022, and submitted to the U.S. Patent and Trademark Office via EFS-Web, entitled 39604WO_9704_02_PC_SequenceListing.txt, is incorporated herein by reference. [Background technology]

[0003] Precise administration of diagnostic or therapeutic agents to the desired site presents an unsolved challenge. Currently, the two main types of gene therapy delivery systems are viral delivery systems and non-viral delivery systems (Non-Patent Document 1; Non-Patent Document 2). Regarding viral vectors, although highly effective, safety is a major concern. Non-viral carriers, including polyplexes and lipoplexes, especially lipid nanoparticles (LNPs), often suffer from a lack of targeting specificity. Recent studies have been increasing interest in extracellular vesicles (EVs) as a promising new class of carriers for RNA drug delivery due to their biocompatibility, intrinsic function of mediating molecular exchange between cells, and natural ability to target specific tissues and cross the blood-brain barrier (BBB) ​​(Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9). However, translation of EV-based therapies to the clinic is hindered by the lack of control over which molecules are loaded into EVs from EV-producing donor cells. Depending on the donor cell type, EV cargo can include proteins, DNA, RNA, lipids, nutrients, and metabolic waste products. Unwanted intracellular components are not excluded from EVs, impeding not only the loading capacity, but also potentially harmful components, such as overexpression constructs introduced to engineer EVs, and cellular waste products, are delivered to the target.

[0004] Although many systems have been developed to load small RNA cargoes, such as siRNA and microRNA, into EVs, active enrichment of long mRNAs in EVs remains a challenge (Non-Patent Document 4). Extremely low copy numbers have been reported for endogenous EV-associated RNAs, ranging from 0.02 to 1 RNA per EV, and small RNAs are packaged into EVs more efficiently than long mRNAs (0.01 to 1 microRNA per EV vs. 0.001 long intact RNA) (Non-Patent Document 10; Non-Patent Document 11; Non-Patent Document 12; Non-Patent Document 13). Only 8% of the mRNAs in donor cells are detected in their EVs (Non-Patent Document 14). Already reported methods of loading mRNA into EVs include active and passive encapsulation (Non-Patent Document 15). For example, to treat Parkinson's disease (PD), catalase mRNA was loaded by incubation with macrophage-derived EVs after sonication and ejection or permeabilization with saponin (Non-Patent Document 6). To treat leukemia, antisense oligonucleotides (ASOs), CRISPR-Cas9 mRNA, and guide RNA (gRNA) were delivered into EVs derived from enucleated red blood cells (RBCs) by electroporation (Non-Patent Document 16). EVs are also engineered at the parent cell level, with genetic components introduced to induce the production of designed EVs, often with gross overexpression of cargo components to achieve sufficient loading dose. These procedures may damage EVs, leading to their aggregation, or may alter the physiology of EV-producing donor cells, subsequently reducing cargo loading (Non-Patent Document 15; Non-Patent Document 17; Non-Patent Document 18).

[0005] The available methods of delivering nucleic acid to cells have well-characterized limitations. For example, AAV viral vectors, which are often used for gene therapy, are immunogenic, have a payload capacity limited to about 4.4 kb, suffer from poor biodistribution, are administered only by direct injection, and present the risk of disrupting host genes through integration. Non-viral methods have different limitations. Liposomes are primarily delivered to the liver. Extracellular vesicles have limited scalability and are difficult to purify. Thus, the need for new methods of delivering therapeutic payloads is recognized.

[0006] Most molecules have no inherent affinity in the body. In other cases, administered drugs accumulate in the liver and kidneys or in unintended tissues or cell types due to clearance. Methods for improving delivery include coating the drug of choice with hydrophobic compounds or polymers. Such approaches increase the persistence of the drug in circulation and enhance its hydrophobicity for intracellular uptake. On the other hand, this approach does not actively direct the cargo to the desired site for delivery.

[0007] In order to specifically target the site where treatment is required, therapeutic compound is optionally fused with a portion such as ligand, antibody and aptamer, which recognizes and binds to the receptor displayed on the surface of targeted cell.After reaching the target cell, therapeutic compound is optionally further delivered to intracellular target.For example, therapeutic RNA is translated into protein when it contacts with ribosome in the cytoplasm of cell. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] YK Sung and SW Kim, Biomater Res 23, 8 (2019) [Non-Patent Document 2] N.Nayerossadatら, Adv Biomed Res 1, 27(2012) [Non-licensed document 3] Tangら、Front Oncol 9, 1208(2019)

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Non-licensed literature 9

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Non-licensed Document 15

[0009] The present disclosure is directed to engineered extracellular vesicles, methods of making engineered extracellular vesicles, and methods of using engineered extracellular vesicles for delivery of therapeutic compounds, biopharmaceuticals, or both, to tissues and cells of interest. [Means for solving the problem]

[0010] In a first aspect, the present disclosure is directed to an RNA transcript composition comprising a cargo mRNA comprising an Arc 5'UTR sequence. In some embodiments, the RNA transcript composition further comprises an Arc mRNA. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence derived from a mammal. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence selected from the group consisting of human, mouse, and rat. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence derived from Drosophila. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 1-4. In some embodiments, the cargo mRNA further comprises a poly(A) signal. In some embodiments, the cargo mRNA encodes a therapeutic protein. In some embodiments, the cargo mRNA encodes a peptide, an enzyme, a cytokine, a hormone, a growth factor, an antigen, an antibody, a portion of an antibody, a clotting factor, a regulatory protein, a signaling protein, a transcription protein, and / or a receptor. In some embodiments, the cargo mRNA encodes a fluorescent protein, a bioluminescent protein, and / or a recombinase reporter. In some embodiments, the Arc mRNA comprises an Arc 3'UTR sequence. In some embodiments, the Arc mRNA comprises an Arc 3'UTR sequence derived from a mammal. In some embodiments, the mammal is a human, a mouse, or a rat. In some embodiments, the Arc 3'UTR sequence comprises an Arc 3'UTR sequence derived from Drosophila. In some embodiments, the Arc 3'UTR sequence comprises an Arc 3'UTR sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 5-8. In some embodiments, the Arc mRNA further comprises a poly(A) signal.In some embodiments, the Arc mRNA encodes an Arc protein derived from a mammal. In some embodiments, the mammal is a human, a mouse, or a rat. In some embodiments, the Arc mRNA encodes an Arc protein derived from Drosophila. In some embodiments, the Arc mRNA comprises an Arc mRNA sequence derived from a mammal. In some embodiments, the mammal is a human, a mouse, or a rat. In some embodiments, the Arc mRNA comprises an Arc mRNA sequence derived from Drosophila. In some embodiments, the Arc mRNA comprises a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 9-12.

[0011] Some aspects of the present disclosure are directed to a recombinant system comprising a DNA encoding a cargo mRNA having an Arc 5'UTR sequence. In some embodiments, the recombinant system comprising a DNA encoding a cargo mRNA having an Arc 5'UTR sequence further comprises a second DNA encoding an Arc mRNA. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence derived from a mammal. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence selected from the group consisting of human, mouse, and rat. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence derived from Drosophila. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 1-4. In some embodiments, the cargo mRNA further comprises a poly(A) signal. In some embodiments, the cargo mRNA encodes a therapeutic protein. In some embodiments, the cargo mRNA encodes a peptide, an enzyme, a cytokine, a hormone, a growth factor, an antigen, an antibody, a portion of an antibody, a clotting factor, a regulatory protein, a signaling protein, a transcription protein, and / or a receptor. In some embodiments, the cargo mRNA encodes a fluorescent protein, a bioluminescent protein, and / or a recombinase reporter. In some embodiments, the Arc mRNA comprises an Arc 3'UTR sequence. In some embodiments, the Arc 3'UTR sequence comprises an Arc 3'UTR sequence derived from a mammal. In some embodiments, the Arc 3'UTR sequence comprises an Arc 3'UTR sequence selected from the group consisting of human, mouse, and rat. In some embodiments, the Arc 3'UTR sequence comprises an Arc 3'UTR sequence derived from Drosophila.In some embodiments, the Arc 3'UTR sequence comprises an Arc 3'UTR sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 5-8. In some embodiments, the Arc mRNA further comprises a poly(A) signal. In some embodiments, the system comprises a single plasmid comprising a DNA encoding a cargo mRNA having an Arc 5'UTR sequence and a second DNA encoding the Arc mRNA. In some embodiments, the system comprises a first plasmid comprising a DNA encoding a cargo mRNA having an Arc 5'UTR sequence; and a second plasmid comprising a second DNA encoding the Arc mRNA. In some embodiments, the plasmid(s) further comprises a heterologous DNA regulatory element. In some embodiments, the heterologous DNA regulatory element comprises a promoter, an enhancer, a silencer, an insulator, or a combination thereof. In some embodiments, the Arc mRNA comprises an Arc sequence derived from a mammal. In some embodiments, the mammal is a human, a mouse, or a rat. In some embodiments, the Arc mRNA comprises an Arc sequence derived from Drosophila. In some embodiments, the Arc 5'UTR sequence comprises a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 1-4. In some embodiments, the Arc mRNA encodes an Arc protein derived from a mammal. In some embodiments, the mammal is a human, a mouse, or a rat. In some embodiments, the Arc mRNA encodes an Arc protein derived from Drosophila. In some embodiments, the Arc mRNA comprises an Arc mRNA derived from a mammal. In some embodiments, the mammal is a human, a mouse, or a rat. In some embodiments, the Arc mRNA comprises an Arc mRNA derived from Drosophila.In some embodiments, the Arc mRNA comprises a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs:9-12.

[0012] Certain aspects of the present disclosure are directed to extracellular vesicles comprising an Arc protein; and a cargo mRNA comprising an Arc 5'UTR sequence. Some embodiments are directed to extracellular vesicles comprising an Arc protein; and a cargo mRNA comprising an Arc 5'UTR sequence, where the Arc 5'UTR sequence comprises an Arc 5'UTR sequence derived from a mammal. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence selected from the group consisting of human, mouse, and rat. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence derived from Drosophila. In some embodiments, the Arc 5'UTR sequence comprises an Arc 5'UTR sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 1-4. In some embodiments, the cargo mRNA further comprises a poly(A) signal. In some embodiments, the cargo mRNA encodes a therapeutic protein. In some embodiments, the cargo mRNA encodes a peptide, an enzyme, a cytokine, a hormone, a growth factor, an antigen, an antibody, a portion of an antibody, a clotting factor, a regulatory protein, a signaling protein, a transcription protein, and / or a receptor. In some embodiments, the cargo mRNA encodes a fluorescent protein, a bioluminescent protein, and / or a recombinase reporter. In some embodiments, the Arc protein comprises an Arc protein sequence derived from a mammal. In some embodiments, the mammal is a human, a mouse, or a rat. In some embodiments, the Arc protein comprises an Arc protein sequence derived from Drosophila melanogaster. In some embodiments, the Arc protein comprises at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 13-16. In some embodiments, the Arc mRNA comprises an Arc 3'UTR sequence.In some embodiments, the Arc 3'UTR sequence comprises an Arc 3'UTR sequence derived from a mammal. In some embodiments, the mammal is a human, a mouse, or a rat. In some embodiments, the Arc 3'UTR sequence comprises an Arc 3'UTR sequence derived from Drosophila. In some embodiments, the Arc 3'UTR sequence comprises an Arc 3'UTR sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 5-8. In some embodiments, the extracellular vesicles further comprise one or more small molecule drugs.

[0013] Another aspect of the present disclosure is a method for producing extracellular vesicles, comprising: (a) obtaining cells comprising Arc mRNA and a cargo mRNA comprising Arc 5'UTR; (b) growing the cells in a medium under conditions for expressing Arc protein encoded by Arc mRNA, wherein the cells produce extracellular vesicles comprising Arc protein and cargo mRNA having Arc 5'UTR sequence; and (c) isolating the extracellular vesicles from the medium. In some embodiments of the method, the cells of step (a) are obtained by introducing into a donor cell a DNA construct transcribed into Arc mRNA and a DNA construct transcribed into cargo mRNA. In some embodiments of the method, the cells of step (a) are obtained by introducing into a donor cell Arc mRNA and cargo mRNA. In some embodiments, the recombinant construct is delivered in the form of DNA, RNA, or a combination of both. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the donor cells are selected from neural cells, epithelial cells, endothelial cells, hematopoietic cells, connective tissue cells, muscle cells, bone cells, chondrocytes, germline cells, adipocytes, stem cells, autologous ex vivo differentiated cells, iPSC-derived ex vivo differentiated cells, cancer cells, and combinations thereof. In some embodiments, the donor cells are white blood cells. In some embodiments, the donor cells are autologous ex vivo differentiated white blood cells. In some embodiments, the donor cells are autologous ex vivo differentiated monocytes, macrophages, dendritic cells, or combinations thereof. In some embodiments, the donor cells are iPSC-derived ex vivo differentiated white blood cells. In some embodiments, the donor cells are iPSC-derived ex vivo differentiated monocytes, macrophages, dendritic cells, or combinations thereof.In some embodiments, cells containing the nucleic acid construct are prepared by transfecting the cells with the nucleic acid construct, where the transfection is performed by polyethyleneimine (PEI) complexation, electroporation, cationic lipid complexation, lipid nanoparticle-mediated delivery, microinjection, and combinations thereof.

[0014] One aspect of the present disclosure is directed to a method for delivering mRNA to a recipient cell, comprising obtaining the extracellular vesicles described; and contacting the recipient cell with the extracellular vesicles, where the extracellular vesicles fuse with the recipient cell, thereby delivering the mRNA to the recipient cell. In some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed in vivo. In some embodiments, the recipient cell is a mammalian cell. In some embodiments, the recipient cell comprises a hematopoietic cell, a non-hematopoietic cell, a stem cell, or a combination thereof. In some embodiments, the mRNA is delivered to the recipient cell to treat a disease, produce a protein, induce cell death, inhibit cell death, alter cellular senescence, induce immune tolerance, modulate an existing immune response, modify intracellular activity, modify cell behavior, or a combination thereof.

[0015] Another aspect of the present disclosure is directed to a method for treating a subject in need thereof, comprising obtaining the extracellular vesicles described; and administering the extracellular vesicles to the subject. In some embodiments, the extracellular vesicles are administered orally, rectally, intravenously, intramuscularly, subcutaneously, intrauterinely, intracerebrovascularly, or intraventricularly. In some embodiments, the extracellular vesicles comprise CRISPR-associated protein mRNA and guide RNA adapted for treating diseases, including genetic disorders. In some embodiments, the extracellular vesicles are administered to a subject for treating neurodegenerative diseases, aging-related disorders, brain tumors, inflammatory conditions, and specifically deliver RNA across the blood-brain barrier to inflammatory brain tissue without affecting healthy cells. In some embodiments, the extracellular vesicles are adapted to deliver APOE4 RNA to the brain for treating Alzheimer's disease. In some embodiments, the extracellular vesicles are administered to treat cancer, and target tumor cells without affecting healthy tissue. In some embodiments, the extracellular vesicles comprise mRNA corresponding to tumor-associated antigens, and the extracellular vesicles are delivered as cancer vaccines for the treatment of cancer, including melanoma, colon cancer, gastrointestinal cancer, genitourinary cancer, and hepatocellular carcinoma. In some embodiments, the extracellular vesicles are delivered for the prevention and / or treatment of infectious diseases. In some embodiments, the extracellular vesicles are delivered for the treatment of autoimmune diseases.

[0016] Another aspect of the present disclosure is directed to a method of delivering constructs to recipient cells in vivo to produce the extracellular vesicles described in vivo using endogenous Arc. In some embodiments, the vesicles are produced in vivo by endogenous Arc. In some embodiments, the constructs are delivered in the form of DNA and / or RNA. In some embodiments, the constructs are delivered by lipid nanoparticles, exosomes, viruses, and other gene delivery methods.

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0018] [Figure 1-1] 1A-1J: Characterization of eaEVs loaded with and transducing mRNA. (A) eaEV production from donor cells and eaEV transduction into recipient cells. (B) Fluorescent NTA measures the concentration and size distribution of fluorescently labeled anti-Arc+ eaEVs among all light scattering by EVs (screen capture shown in C: red circles, eaEVs; green circles, non-Arc EVs). (D) The size of each particle was plotted as a function of its scattering intensity. (E) The size distribution profile by NTA is represented as a histogram of particle concentration. (F) The percentage of eaEVs in total EVs increased with Arc mRNA transfection, although total EV production was also promoted by liposomal transfection alone. (G) Representative NSEM images of eaEVs with and without an outer membrane. (H) Fluorescence intensity of total EVs (CMDR+) and eaEVs (anti-Arc+) was measured to quantify their concentration after purification. The numbers obtained from this fast and easy assay were then correlated with the NTA results to calculate the absolute particle number in each sample for future reference. (I) Confocal microscopy observed the release of exosomes via membrane fusion of multivesicular bodies (MVBs), direct extracellular budding, and / or endocytosis of EVs in donor cell cultures (blue pixels highlight Arc protein within EVs, whereas green pixels label A5U-GFP cargo mRNA). (J) Overlap of GFP with CMDR in recipient cells: DAPI+ / CMDR- cells without EV uptake show no GFP signal. [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 1-5] Continued from Figure 1-4. [Figure 1-6] Continued from Figure 1-5. [Figure 1-7] Continued from Figure 1-6. [Figure 1-8] Continued from Figure 1-7. [Figure 2-1]2A-2Q show that the addition of A5U significantly improved the efficiency of packaging of mRNA into eaEVs. (A) The 5'UTR of rat Arc shows a higher similarity in predicted secondary structure to the 5'UTR of HIV1 than the 5'UTRs of mouse and human Arc. The amount of Cy3-A5U-GFP mRNA or Cy5-GFP mRNA loaded into EVs was increased in the presence of Arc capsids, as shown by fluorescence intensity readings for purified EVs (B and E) and real-time epifluorescence imaging of donor RAW264.7 cell cultures (C-D and F-G). (H) RT-qPCR following RIP revealed that the addition of A5U increased the loading of mRNA into Arc capsids. (I-J) Optimization of transfection reagent ratios allows efficient and selective loading of A5U-GFP cargo compared to 18S, GAPDH, and rArc cargo. Recipient cells were stained and imaged for A5U-GFP mRNA cargo, Arc capsid mRNA, Arc capsid protein, and nuclei with 614 total cells and 1611 analyzed EVs. Representative areas are shown in (K) and enlarged views in (L, M-O). Circles highlight Arc protein, indicating individual eaEVs. At a ratio of Arc:A5U-GFP=1.5:1, nearly all Arc(protein)+EVs overlapped with A5U-GFP mRNA (M-O, green and yellow), but no Arc(protein)+EVs overlapped with Arc mRNA (M-O, red). In addition to being loaded into Arc EVs, A5U-GFP and Arc mRNA are also found in other EVs (L, red and yellow). GFP protein is packaged into EVs at a very low frequency compared to mRNA cargo (L, MO, purple). The percentage of colocalization of capsid protein with GFP mRNA was quantified and compared to Arc mRNA (P-Q). No overlap of Arc protein with Arc mRNA was observed in extracellular vesicles. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Figure 2-5] Continued from Figure 2-4. [Figure 2-6] Continued from Figure 2-5. [Figure 3-1] Figures 3A-3G show that the addition of A5U significantly improved the efficiency and stability of mRNA delivery to recipient cells. (A1-A2) Real-time live cell imaging of Cy3 fluorescence was performed to quantify cargo mRNA taken up by recipient cells at 15 min (B1-B4), 1 h, 4 h (C1-C4), 3 days, 6 days, and 12 days after EV transduction. Transduction with eaEV carrying A5U-GFP showed a highly stable and significant increase in the amount of cargo accumulated in recipient cells (A1), whereas Arc EV carrying cargo without A5U appeared to be less stable and less efficient (A2). (D) First, CMDR (cells are dark red) membrane dye was used to label total EVs, allowing for rapid and accurate quantification of total EVs in buffer as well as in recipient cells. Titration experiments suggested the best dye concentration, 1:5000, for moderate fluorescence signals, making individual EVs visible in recipient cells. This dye concentration was then used to optimize the amount of EVs introduced into recipient cells, confirming a linear correlation between CMDR signal and EV concentration. (E) We added the same amount of EVs from each group to recipient cells and calculated their CMDR fluorescence. After 2 hours, recipient cells treated with EVs from different groups showed similar CMDR fluorescence levels, suggesting uptake of a similar number of total EVs. (F) Arc / A5U-GFP showed a consistent increase in cargo translation between the control groups at 4 hours and 1 day after EV transduction. At day 3, in the control group, recipient cells began to show strong autofluorescence, and the increase with Arc / A5U-GFP was no longer significant. (G) The percentage of recipient cells expressing GFP was low but was significantly increased by Arc / A5U GFP. [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Diagram 3-4] Continued from Figure 3-3. [Figure 3-5] Continued from Figure 3-4. [Diagram 3-6] Continued from Figure 3-5. [Figure 4-1]4A-4J: A5U-eaEV enriches cargo mRNA in the aging brain. (A) BM cells were extracted from mouse femurs and cultured in complete medium supplemented with GM-CSF and IL4 for 7 days to differentiate into monocytes, dendritic cells, and macrophages, from which control and engineered EVs were produced and injected intravenously into a mouse model of neuroinflammation. (B) Light-colored file image of representative GM-CSF / IL4BM culture morphology at day 6. (C) Phenotype of representative GM-CSF / IL4BM culture at day 6. CD11c+MHCII+ BMDCs (C, top left) are subdivided based on CD11b and MHCII expression (C, bottom). Boxes depict gates and numbers correspond to the percentage of cells within each gate. Histograms showing surface expression of markers indicative of MHCIIhighCD11blow and MHCIIintCD11bhigh subsets. (D) GM-CSF / IL4 BM cells at day 6 were able to take up their own EVs labeled with CMDR. After transfection of (E1-E4) mRNA transcripts, EVs were produced for 40 hours before harvesting and purification. This extended production resulted in saturation of EVs in the supernatant culture medium, resulting in approximately equal numbers of total EVs from each sample group (F). Meanwhile, the proportion of eaEVs in total EVs was significantly higher in the Arc / A5U-GFP group. Purified EVs (1x and 2x dilutions) were stained with CMDR and fluorescent Arc antibodies, and their epifluorescence intensity was measured for EV concentration. (G) Representative IVIS images show in vivo biodistribution of Cy3+ A5U-GFP mRNA, which was significantly enriched in the aged brain with systemic administration of eaEV compared to the no Arc A5U-GFP control alone. Photo overlays are presented for radiance with a color range of 3.3e+7 to 4.9e+8 and a color threshold of 3.5e+8, based on negative control animals, to subtract background signal.In addition to the representative images shown in this figure, mock transfection controls, PBS (no EV) injected controls, and CMDR+ animals were also analyzed as negative controls for Cy3 IVIS imaging in this figure. (H) IVIS imaging of in vivo biodistribution of Cy5+GFP mRNA suggests that mRNA cargo is not enriched in the aging brain by eaEV delivery without A5U motifs added to the cargo construct. As in (G), a color threshold of 3.5e+8 was applied to subtract background signal based on negative control animals (Cy3+ Cy5-Arc / A5U-GFP and A5U-GFP groups). (I-J) Quantification of IVIS signal. Mean ± SD, n=2 for experimental groups; n=7 for NC group. A sample size of 6 per group was used based on power analysis with a power of 80%, a significance level of 5%, and a two-tailed t-test following Cohen's power analysis. [Figure 4-2] Continued from Figure 4-1. [Figure 4-3] Continued from Figure 4-2. [Figure 4-4] Continued from Figure 4-3. [Figure 4-5] Continued from Figure 4-4. [Figure 4-6] Continued from Figure 4-5. [Figure 5-1]Figure 5A-5G shows that BM-DC / M-derived A5U-eaEVs can deliver mRNA to neurons across the BBB to target chronic pan-neuronal inflammation. (A, A', B, B'; where A' and B' are enlargements of A and B) A5U-GFP mRNA was successfully delivered across the BBB to express GFP protein in the inflamed aged brain: white pixels (GFP+ / NeuN+) highlight the colocalization between GFP and NeuN-Alexa647, representing GFP expression in neurons. In contrast, green pixels showed GFP in non-neuronal cells, likely infiltrating immune cells. Enlarged views of the hypothalamus are shown in (C-D). (E-F) Two days after IV injection, and another six days later, the aged brain showed a significant increase in GFP expression levels in NeuN+ cells. Systemic injection of eaEV / A5U-GFP resulted in comparable levels of GFP expression in infiltrating peripheral immune cells (green pixels, C-E), but significant enrichment of NeuN+ neurons in the aging brain (white pixels, C-E). (G) Integrated expression of GFP between control and experimental groups in various brain regions. Certain brain regions, including the arcuate nucleus of the hypothalamus (ARH), medial preoptic nucleus (MPN), and ventral tegmental area (VTA), showed more significant increases than other regions, including the hippocampus and prefrontal cortex (PFC). [Figure 5-2] Continued from Figure 5-1. [Figure 5-3] Continued from Figure 5-2. [Figure 5-4] Continued from Figure 5-3. [Figure 5-5] Continued from Figure 5-4. [Figure 6-1]Figures 6A-6H show that BM-DC / M-derived A5U-eaEVs can deliver mRNA to neurons across the BBB and target injury from acute ischemic stroke. In the acute ischemic stroke model (A), eaEVs specifically delivered GFP to neurons in the stroke area (B-C) without affecting the control area (B-C'). Further enlargements (D-E) in (F-G) show GFP expression in many NeuN+ neurons and in a few Iba1+ microglia / macrophages (white arrows), which also showed many NeuN+ / Iba1+ cells without GFP expression, suggesting that the observed GFP signal is specific. It is clear that GFP expression is also seen in NeuN- / Iba1- cells. (H) In the stroke area, the number of NeuN+ neurons was decreased, whereas the number of Iba1+ immune cells (microglia and infiltrating macrophages) was increased, compared to the control area. The number of GFP-expressing cells was increased. All GFP+ cell counts in the control area are non-specific background signals, originating from autofluorescence in microvessels. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 6-4] Continued from Figure 6-3. [Figure 6-5] Continued from Figure 6-4. [Figure 7-1]7A-7E show the transfection of engineered DNA constructs and RNA transcripts into donor cells to investigate their functionality. (A) Cargo-encoding DNA constructs and RNA transcripts validated in HEK293 and RAW 264.7 cells with random mutation negative control, significantly different fluorophore mCherry control, and mock transfection control by live cell epifluorescence imaging applied to monitor expression in real time time course. (B) Expression by RAW264.7 donor cells 8 hours after transfection. (C) Expression by RAW264.7 donor cells 24 hours after transfection. (D-E) Donor cell numbers and viability at the end of EV production were recorded and compared at all times to ensure good and comparable quality of EVs produced between control and experimental groups. [Figure 7-2] Continued from Figure 7-1. [Figure 7-3] Continued from Figure 7-2. [Figure 7-4] Continued from Figure 7-3. [Figure 7-5] Continued from Figure 7-4. [Figure 8-1] Figures 8A-8C show the optimization of RNA transfection and EV production titration by real-time live cell imaging. (A) Real-time live cell imaging of donor cells transfected at six doses of Lipofectamine. (B) Graphical representation of cell numbers in a 96-well plate at 4 hours post-transfection. (C) Comparison of cell numbers in a 6-well plate at 24 hours post-transfection and 96 hours post-transfection. The conclusion is that too much mRNA without enough Lipofectamine reduced donor cell viability. The optimal dose was determined to be 100 ng transfected mRNA per 20,000 donor cells (96-well; 100 μL total opti-MEM medium with 0.3 μL Lipofectamine per well). [Figure 8-2]Continued from Figure 8-1. [Figure 8-3] Continued from Figure 8-2. [Figure 9-1] 9A-9B show the optimization of the ratio of the transfection components capsid and cargo mRNA. (A) Detailed optimization of the transfection ratio between capsid and cargo mRNA was performed in RAW264.7 cells. The lowest ratio of Arc:GFP is 0:0 is shown in (A1), while the ratio of Arc:GFP is 3:3 is shown in (A16). (B) The graph shows that increasing the amount of capsid Arc mRNA transfected resulted in a decrease in GFP protein expression, since more cargo A5U-GFP mRNA was not translated and was encapsulated. It was decided not only to focus on the amount of cargo mRNA that resulted in a high level of GFP expression when transfected alone, but also to focus on the significant decrease in GFP expression when capsid mRNA was introduced compared to having cargo alone. The ratio determined was 3:2 Arc:A5U-GFP (or 1.5:1 Arc:A5U-GFP), shown in A12. [Figure 9-2] Continued from Figure 9-1. [Figure 10] Figure 1. Visualization and optimization of RNA encapsulation: (A) Real-time live cell imaging of transfected cells using Cy3 and Cy5. (B) Graphical representation of fluorescence intensity readings for RNA encapsulation. [Figure 11-1] Figure 1 shows the time course of DNA transfection with quantification of GFP expression. For DNA transfection with PEI into HEK293 cells, since there is a stable source for the continuous production of both capsid and cargo mRNA and protein, we observed a significant and stable increase in GFP expression transfecting both cargo and capsid. Eventually, GFP expression in donor cells reached saturation (by 24 hours). [Figure 11-2] Continued from Figure 11-1. [Figure 12-1]Figures 12A-12M show EV characterization and optimization of EV production. (A-C) NTA results suggested that total EV production was comparable among all transfected control and experimental groups. (K-M) Transfection of Arc produced larger vesicles, likely the production of Arc ectosomes. (D-H) Addition of Arc capsid and A5U-GFP cargo allowed the most significant secretion of Arc EVs compared to other control groups, including Arc / GFP. (H) A large proportion of EVs among all EVs in Arc / A5U-GFP were Arc ectosomes. (I) Serum-free production of EVs resulted in the secretion of high levels of mouse CD63+ EVs and a large proportion of eaEVs among all EVs secreted. (J) EVs can be stored for short periods at 4°C, but then began to aggregate. [Figure 12-2] Continued from Figure 12-1. [Figure 12-3] Continued from Figure 12-2. [Figure 12-4] Continued from Figure 12-3. [Figure 12-5] Continued from Figure 12-4. [Figure 12-6] Continued from Figure 12-5. [Figure 12-7] Continued from Figure 12-6. [Figure 13] Figure 1 shows the optimized CMDR dye used for quantification of EV uptake. Staining with CMDR, a cell membrane dye, was performed to facilitate quantification of EVs in recipient cells. The number of EVs transferred into recipient cells was also optimized. [Figure 14A] Figure 2 shows the biodistribution of total EVs (CMDR+) at 3 days after IV injection. When the total number of EVs (per kg body weight) injected into each control or test mouse was the same, the biodistribution of total EVs (mostly degraded) was similar in all harvested organs at 3 days after IV injection. Total EVs were labeled with CMDR, a cell membrane dye. [Figure 14B]Figure 2 shows the biodistribution of total EVs (CMDR+) at 3 days after IV injection. When the total number of EVs (per kg body weight) injected into each control or test mouse was the same, the biodistribution of total EVs (mostly degraded) was similar in all harvested organs at 3 days after IV injection. Total EVs were labeled with CMDR, a cell membrane dye. [Figure 15-1] 15A-15E show that eaEVs can deliver mRNA to tumors. (A) In vivo biodistribution of CMDR+ total EVs in control and experimental groups: (1) no EV negative control (NC); (2) mock transfected (noTrans) EV control; (3) Arc-negative EVs carrying GFP mRNA or A5U-GFP mRNA; (4) Arc-positive EVs loaded with GFP mRNA or A5U-GFP mRNA. Photo overlay of radiance with color range of 3.3e+7-5.0e+9 and threshold of 3.5e+8. (B) Quantification of CMDR biodistribution in mice injected with leukocyte eaEVs. (C) High-resolution CLSM images showed high levels of GFP expression in tumors but minimal expression in other organs. Whole tumor imaging (D–E) with K-Ras staining (D1–E1) and close-up views of deep tumor regions, far from large blood vessels (D2–E3), suggest that eaEVs facilitate deep tumor penetration and delivery of mRNA. [Figure 15-2] Continued from Figure 15-1. [Figure 15-3] Continued from Figure 15-2. [Figure 15-4] Continued from Figure 15-3. [Figure 16] Figure 1 shows that eaEVs can load small molecule drugs into MB231 triple-negative breast cancer cells. When SM drug 1 was loaded into eaEV-producing donor cells at a concentration of 1:500, these eaEVs were able to deliver the fluorescently labeled drug into recipient cells (top), whereas the drug loaded at 1:50000 was too diluted to introduce the drug as a negative control (bottom). [Figure 17] FIG. 1 shows maps for exemplary DNA plasmids used to prepare capsids for engineered EVs. [Figure 18] 18A-18B show the mechanism of selective cargo loading and the structure of an exemplary carrier. (A) The Arc 5'UTR allows the recognition of specific cargo mRNA by the Arc capsid protein, while the Arc 3'UTR accelerates nonsense-mediated mRNA decay of the Arc capsid mRNA after its translation. Taken together, these allow selective loading of cargo without interference of overexpressed capsid mRNA. (B) An exemplary structure of an engineered EV containing the Arc protein capsid and nucleic acid cargo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Extracellular vesicles (EVs), natural nanocarriers, are a promising new class of drug carriers due to their biocompatible nature and endogenous function of mediating long-range intercellular exchange of molecules with natural ability to deliver to desired targets. In contrast, therapeutic messenger RNA (mRNA) has attracted increasing interest in recent years. However, efficient and selective encapsulation of long mRNA into EVs remains problematic. Disclosed herein is a virus-like, but retrotransposon, Arc protein capsid incorporated into the lumen of EVs ("Arc EVs"). Arc EVs possess high efficacy and biocompatibility as naturally occurring vesicles, similar to viral vectors. Arc EVs also play a natural role in loading and interneuronal transport of mRNA, making them an important tool for delivery of mRNA to the brain, among other target tissues and cells of interest. The disclosed engineered Arc EV (eaEV) further allows highly efficient and stable encapsulation of specific mRNA cargo. Naturally equipped with homing molecules to the neuroinflammatory microenvironment by donor cells, leukocyte-derived eaEV facilitates efficient delivery of mRNA to diseased neurons across the blood-brain barrier. The present disclosure presents a novel endogenous virus-like system that can load and deliver specific mRNA to target tissues and cells of interest.

[0020] Enabled by the incorporation of a virus-like protein capsid that binds to RNA motifs contained within the cargo mRNA, the engineered Arc EVs have a high mRNA cargo loading / transduction efficiency. The immunologically inert eaEVs are produced from diverse cell types, including monocyte-derived cells, to deliver mRNA across the blood-brain barrier (BBB) ​​and specifically target the neuroinflammatory microenvironment in vivo, supporting the potential therapeutic potential of this nanoscale, biocompatible, and efficient mRNA drug carrier.

[0021] Extracellular vesicles As used herein, the term "extracellular vesicles (EVs)" or "vesicles" refers to cell-derived vesicles resulting from a combination of endocytic and exocytic events that result in the encapsulation of a variety of biomolecules. All prokaryotic and eukaryotic cells release EVs as part of their normal physiology and during acquired disorders. EVs are divided into two categories, ectosomes and exosomes, but for the purposes of this disclosure, the terms "ectosomes," "exosomes," and "EVs" are used interchangeably. Ectosomes are vesicles that are pinched off from the surface of the cell membrane via extracellular budding and include microvesicles, microparticles, and large vesicles with sizes ranging from about 50 nm to 1 μm in diameter. Exosomes are EVs derived from endosomes with sizes ranging from about 40 to 160 nm in diameter (average about 100 nm). Such encapsulation may protect the therapeutic nucleic acid from enzymatic degradation or other environmental stresses (e.g., ionic strength, pH, etc.) Protein association with EVs provides stability in both extracellular and intracellular environments as well as facilitating a cellular targeting mechanism for intercellular communication.

[0022] In some embodiments, EVs are created in prokaryotes, eukaryotes, or viruses. In some embodiments, engineered EVs are made in yeast, bacteria, viruses, protists, or other types of cells, whether they are unicellular organisms, multicellular organisms, or non-organisms that contain DNA.

[0023] Exosomes are produced by many different types of cells, including immune cells such as B lymphocytes, T lymphocytes, dendritic cells (DCs), and mast cells. Exosomes are also produced by, for example, glioblastoma cells, platelets, reticulocytes, neurons, intestinal epithelial cells, and tumor cells. Exosomes for use in the disclosed compositions and methods can be derived from any suitable cell, including the cells identified above. Exosomes have also been isolated from body fluids such as plasma, urine, amniotic fluid, and malignant tumor effusions. Non-limiting examples of exosome-producing cells suitable for large-scale production include dendritic cells (e.g., immature dendritic cells), human embryonic kidney 293 (HEK) cells, 293T cells, Chinese hamster ovary (CHO) cells, and human ESC-derived mesenchymal stem cells.

[0024] In some embodiments, the exosomes are derived from DCs, such as immature DCs. Exosomes produced from immature DCs do not express MHC-II, MHC-I, or CD86. Thus, these exosomes do not significantly stimulate naive T cells and fail to induce responses in mixed lymphocyte reactions, making exosomes produced from immature dendritic cells good candidates for use in the delivery of genetic material.

[0025] Exosomes can also be derived from any autologous patient-derived cells, heterologous haplotype-matched cells, or xenogeneic stem cells, so as to reduce or avoid the occurrence of an immune response in the patient to whom the exosomes are delivered. Any exosome-producing cell can be used for this purpose.

[0026] Exosomes produced by cells are collected from the culture medium by any suitable method. Typically, exosome preparations are prepared from cell culture or tissue supernatants by centrifugation, filtration, or a combination of these methods. For example, exosomes can be collected by centrifugation using a low speed ( Differential centrifugation, which is a <20,000 g centrifugation, followed by high speed (>The pellet may be prepared by centrifugation at 100,000 g, size filtration through a suitable filter (e.g., a 0.22 μm filter), gradient ultracentrifugation (e.g., through a sucrose gradient), or a combination of these methods.

[0027] The disclosed exosomes are administered to a subject by any suitable means. The administration to a human or animal subject is selected from parenteral, intramuscular, intracerebral, intravascular, subcutaneous, or transdermal administration. In some embodiments, the delivery method is delivery by injection. Preferably, the injection is intramuscular or intravascular (e.g., intravenous) injection. A physician will be able to determine the route of administration for each patient who needs treatment.

[0028] Exosomes are preferably delivered as a composition. The composition is formulated for parenteral, intramuscular, intracerebral, intravascular (including intravenous), subcutaneous, or transdermal administration. Compositions for parenteral administration may also include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives. Exosomes are formulated in pharmaceutical compositions, which may include, in addition to exosomes, pharma- ceutically acceptable carriers, thickeners, diluents, buffers, preservatives, and other pharma-ceutically acceptable carriers or excipients, and the like.

[0029] In some embodiments of the present disclosure, a representative composition of engineered EVs includes cellular membrane components (e.g., structural lipids and membrane proteins), Arc proteins or protein motifs (e.g., MA or CA domains of Arc), enriched RNA, and potential other cargo components (e.g., proteins, RNA, DNA, nutrients, metabolites, and bioactive compounds). Engineered Arc EVs enrich for the desired cargo RNA and minimize packaging of undesired cellular components.

[0030] Some embodiments of the present disclosure include an extracellular vesicle composition comprising an Arc protein and a cargo mRNA comprising an Arc 5'UTR sequence.

[0031] In some embodiments of the extracellular vesicle composition, the Arc protein binds to the Arc 5'UTR sequence of the cargo mRNA. The binding facilitates packaging of the cargo mRNA into exosomes. In some embodiments, the Arc 5'UTR sequence improves loading of the cargo mRNA into EVs, and the loading is improved by at least 25%. In some embodiments, the Arc 5'UTR sequence improves loading of the cargo mRNA into EVs, and the loading is improved by at least 50%. In some embodiments, the Arc 5'UTR sequence improves loading of the cargo mRNA into EVs, and the loading is improved by at least 75%. In some embodiments, the Arc 5'UTR sequence improves loading of the cargo mRNA into EVs, and the loading is improved by at least 1000%. In some embodiments, the Arc 5'UTR sequence improves loading of the cargo mRNA into EVs, and the loading is improved by at least 150%. In some embodiments, the Arc 5'UTR sequence improves loading of cargo mRNA into EVs, where loading is improved by at least 200%. In some embodiments, the Arc 5'UTR sequence improves loading of cargo mRNA into EVs, where loading is improved by at least 250%. In some embodiments, the Arc 5'UTR sequence improves transduction of recipient cells by RNA. In some embodiments, the Arc 5'UTR sequence improves transduction of recipient cells by RNA, where transduction is improved by at least 25% relative to cargo mRNA without the Arc 5'UTR sequence. In some embodiments, the Arc 5'UTR sequence improves transduction of recipient cells by RNA, where transduction is improved by at least 50%. In some embodiments, the Arc 5'UTR sequence improves transduction of recipient cells by RNA, where transduction is improved by at least 75%. In some embodiments, the Arc 5'UTR sequence improves transduction of recipient cells by RNA, where transduction is improved by at least 100%.In some embodiments, the Arc 5'UTR sequence improves transduction of recipient cells by RNA, where transduction is improved by at least 125%. In some embodiments, the Arc 5'UTR sequence improves transduction of recipient cells by RNA, where transduction is improved by at least 150%.

[0032] In some embodiments, the EV composition further comprises one or more small molecules. In some embodiments, the EVs are immersed in a drug solution that contains the desired small molecule. In some embodiments, the drug solution contains the desired small molecule at a predetermined concentration. The EVs then take up the small molecule in a passive transport process. In some embodiments, the small molecule is transferred into the EVs via physical means, such as sonication, where the membrane of the EV is manipulated to allow the small molecule to enter the lumen of the EV.

[0033] "Small molecule" refers to a low molecular weight organic compound that can regulate biological processes. Small molecules typically have a molecular weight of at least 100g per mole, 200g per mole, or 500g per mole, 1000g per mole, or 2000g per mole, and no more than 5,000g per mole, 10,000g per mole, 20,000g per mole, 50,000g per mole, or 100,000g per mole (e.g., 100-50,000g per mole, 100-10,000g per mole). Many pharmaceuticals are small molecules and are called small molecule drugs. As used herein, small molecules and small molecule drugs are used interchangeably.

[0034] Some examples of small molecules are insulin, aspirin, and antihistamines. In some embodiments, small molecules include biomolecules such as fatty acids, glucose, amino acids, and cholesterol, as well as secondary metabolites such as lipids, glycosides, alkaloids, and natural phenols. In some embodiments, small molecules are used to treat neurological diseases. In some embodiments, small molecules are used to treat autoimmune disorders. In some embodiments, small molecules are chemotherapeutic or anticancer drugs. In some embodiments, small molecules are inhibitors that target tyrosine kinase cell surface receptors or intracellular serine / threonine kinases involved in intracellular signaling pathways such as PI3K / Akt / mTOR signaling. In some embodiments, small molecules are inhibitors that target apoptotic proteins, epigenetic regulators, and other proteins that deregulate cancer cell proliferation.

[0035] Arc Arc (activity-regulated cytoskeleton-associated protein) regulates endocytic trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. Arc activity is linked to synaptic strength and neuronal plasticity. In experimental mouse models, the phenotype of Arc loss included deficits in long-term memory formation and reduced neuronal activity and plasticity.

[0036] Arc exhibits molecular properties similar to retroviral Gag proteins. A structural / functional relationship appears to exist between Arc and retroviral Gag polyproteins. Arc was identified in a computational search for domesticated retrotransposons that possess Gag-like protein domains. Arc contains structural elements found within viral group-specific antigen (Gag) polyproteins that may have been derived from the Ty3 / gypsy retrotransposon family (Campillos et al., Trends Genet., 22:585-589, 2006; Shepherd, Semin. Cell Dev. Biol., 77, 73-78, 2018; Zhang et al., Neuron, 86, 490-500, 2015). Biochemical studies have shown that mammalian Arc has a positively charged N-terminal domain (NTD) and a negatively charged C-terminal domain (CTD) separated by a flexible linker (Myrum et al., Biochem. J., 468, 2015). Crystal structure analysis of the isolated CTD revealed two lobules, both with significant three-dimensional homology to the capsid (CA) domain of HIV Gag (Zhang et al., 2015). In retroviruses, self-association of the CA allows the assembly of the Gag polyprotein into an immature capsid shell (Lingappa et al., Virus Res., 193, 89-107, 2014; Perilla and Gronenborn, Trends Biochem. Sci., 41, 410-420, 2016). Remarkably, recombinant Arc from Drosophila and rat was subsequently shown to self-assemble into spheroid particles similar to HIV Gag capsids (Ashley et al., 2018; Pastuzyn et al., Cell, 172, 275-288.e18, 2018). Arc capsids are released into extracellular vesicles that can deliver RNA cargo to recipient cells (Ashley et al., Cell, 172, 262-274, 2018; Pastuzyn et al., 2018).These studies implicate Arc as an endogenous neuronal retrovirus, and oligomeric organization of Arc into virus-like capsids mediates RNA capture and cell-to-cell transfer (Parrish and Tomonaga, Cell, 172, 8-10, 2018; Shepherd, 2018).

[0037] In some embodiments, the Arc is a non-human Arc polypeptide. In some embodiments, the Arc polypeptide comprises a full-length Arc polypeptide (e.g., a full-length non-human Arc polypeptide). In other embodiments, the Arc polypeptide comprises a fragment of a non-human Arc, such as a truncated Arc polypeptide, that participates in capsid formation. In further embodiments, the Arc polypeptide comprises one or more domains of a non-human Arc polypeptide, at least one of the domains participates in capsid formation. In further embodiments, the Arc polypeptide is a recombinant Arc polypeptide.

[0038] In some embodiments, the Arc polypeptide is a human Arc polypeptide, at least the RNA-binding domain of which has been modified to bind a cargo that is not natural in the context of human Arc. In some embodiments, the Arc polypeptide comprises a full-length human Arc polypeptide, at least the RNA-binding domain of which has been modified to bind a cargo that is not natural in the context of human Arc protein. In other embodiments, the Arc polypeptide comprises a human Arc fragment, at least the RNA-binding domain of which comprises a modification(s) in its RNA-binding domain. In further embodiments, the Arc polypeptide comprises one or more domains of a human Arc polypeptide, at least one of which participates in the formation of a capsid, and the RNA-binding domain of which has been modified to bind a cargo that is not bound by the native human Arc protein. In further embodiments, the Arc polypeptide is a recombinant human Arc polypeptide, at least the RNA-binding domain of which has been modified to allow loading of a cargo that is not natural in the context of human Arc protein.

[0039] Various domains of Arc polypeptides have been described in the art. For example, highly conserved unique orthologues of mouse Arc gene were identified across tetrapods (mammals, birds, reptiles, amphibians), see Pastuzyn et al., Cell, 172, 275-288.e18, 2018; or Hallin et al., Biochemistry And Biophysics Reports, 26, 100975, 2021. For example, the domain of human Arc polypeptide that participates in capsid formation includes amino acids 205-364 of human Arc polypeptide, such as human Arc polypeptide reported in GenBank under Accession No. 23237 (SEQ ID NO: 13). Arc polypeptides from other species, such as mammalian species, can also be utilized.

[0040] In some embodiments, the Arc polypeptide comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the Arc polypeptide comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the Arc polypeptide comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 14. In some embodiments, the Arc polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the Arc polypeptide comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 15. In some embodiments, the Arc polypeptide comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the Arc polypeptide comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 16. In some embodiments, the Arc polypeptide comprises the amino acid sequence of SEQ ID NO:16.

[0041] Arc capsid Arc monomers oligomerize into virus-like capsids. Arc spontaneously forms oligomeric structures that resemble virus-like capsids. Purified preparations of rat Arc capsids exhibited double-shelled structures with a mean diameter of 32 ± 0.2 nm (Pastuzyn et al., Cell, 172, 275-288e218 (2018)). Similarly, the bacterially expressed and purified Drosophila homolog of Arc, dArc1, also self-assembles into capsid-like structures. Purified Arc protein expressed in an insect cell expression system also assembles into similar virus-like capsids, all of which indicates that Arc oligomerization is not an artifact of bacterial expression. Immature retroviral capsids are formed by uncleaved Gag polyproteins, with the main stabilizing interactions provided by the C-terminal domain (CTD) of the CA region (Mattei et al., Science, 354, 1434-1437 (2016)). The Drosophila homolog of Arc exhibited virus-like behavior, and the self-assembled structure of the homolog closely matched that of HIV-1 and Ty3 capsids (Erlendsson et al., Nat Neurosci., 23, 172-175 (2020)). Both Drosophila Arc homologs formed pentamers and hexamers that together framed the capsid shell, consistent with other viral capsids. The homologs also formed protrusions on the surface of the capsid (Budnik and Thomson, Nature Neuroscience). Arc protein capsids naturally condense mRNA into the lumen of EVs and prioritize the loading of mRNA over other intracellular components, such as DNA, proteins, and metabolic waste products. Arc proteins oligomerize to form capsids that encapsulate Arc mRNA. In the absence of endogenous Arc mRNA, Arc protein capsids can package and transport numerous other RNAs (Pastuzyn et al., Cell, 172, 275-288e218 (2018)).

[0042] Arc mRNA "Arc mRNA" refers to an mRNA that encodes an Arc polypeptide as described herein. In some embodiments, the Arc mRNA comprises a 5' untranslated region (UTR), which is the Arc 5'UTR. In some embodiments, the Arc mRNA comprises a 3' UTR, which is the Arc 3'UTR. In some embodiments, the Arc mRNA is chimeric in that it comprises the Arc 5'UTR, the Arc mRNA coding sequence encoding an Arc polypeptide, and the Arc 3'UTR, two or all of the three sequences are heterologous, i.e., from different species. In some embodiments, the Arc mRNA comprises the Arc 5'UTR, the Arc mRNA coding sequence encoding an Arc polypeptide, and the Arc 3'UTR, all from the same species.

[0043] In some embodiments, the Arc mRNA does not include a 5'UTR, such as the Arc 5'UTR. In some embodiments, the Arc mRNA includes a 5'UTR. In some embodiments, the Arc mRNA includes a 5'UTR that is not the Arc 5'UTR. In some embodiments, the Arc mRNA does not include a 5'UTR.

[0044] In some embodiments, the Arc mRNA does not include a 3'UTR, such as the Arc 3'UTR. In some embodiments, the Arc mRNA includes a 3'UTR, such as the Arc 3'UTR. In some embodiments, the Arc mRNA includes a 3'UTR that is not an Arc 3'UTR sequence. In some embodiments, the Arc mRNA does not include a 3'UTR.

[0045] In some embodiments, the Arc mRNA is an mRNA that encodes an Arc polypeptide derived from a mammal. In some embodiments, the Arc mRNA is an mRNA that encodes a human Arc polypeptide. In some embodiments, the Arc mRNA is an mRNA that encodes a non-human Arc polypeptide. In some embodiments, the Arc mRNA is an mRNA that encodes a mouse Arc polypeptide. In some embodiments, the Arc mRNA is an mRNA that encodes a rat Arc polypeptide.

[0046] In some embodiments, the Arc mRNA is an mRNA encoding an Arc polypeptide from a non-mammalian source, hi some embodiments, the Arc mRNA is an mRNA encoding a Drosophila Arc polypeptide.

[0047] In some embodiments, the Arc mRNA comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:9. In some embodiments, the Arc mRNA comprises a nucleic acid of SEQ ID NO:9. In some embodiments, the Arc mRNA comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:10. In some embodiments, the Arc mRNA comprises a nucleic acid of SEQ ID NO:10. In some embodiments, the Arc mRNA comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:11. In some embodiments, the Arc mRNA comprises a nucleic acid of SEQ ID NO:11. In some embodiments, the Arc mRNA comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 12. In some embodiments, the Arc mRNA comprises the nucleic acid of SEQ ID NO: 12.

[0048] In some embodiments, the Arc mRNA comprises a poly(adenylation) signal.

[0049] In some embodiments, the Arc mRNA comprises an Arc 3'UTR sequence.

[0050] Arc 5'UTR It has been reported that Arc and Gag show little specificity for a particular mRNA in vitro without their 5' untranslated regions (UTRs) (Ashley et al., Cell, 172, 262-274, e211 (2018); Comas-Garcia et al., Viruses, 8, (2016)). As used herein, "Arc 5'UTR (A5U)" refers to the 5'UTR of naturally occurring Arc mRNA. This is the region that is not translated into protein.

[0051] As described above, in some embodiments, the 5'UTR is optional for the Arc mRNA. For example, in some embodiments, the Arc mRNA includes a 5'UTR; in other embodiments, the Arc mRNA does not include a 5'UTR; in other embodiments, the Arc mRNA includes a 5'UTR that is not the Arc 5'UTR; in other embodiments, the Arc mRNA does not include a 5'UTR at all.

[0052] In the embodiment in which Arc mRNA comprises A5U, A5U is an A5U sequence derived from a mammal. In further embodiments, A5U is derived from a human. In other embodiments, A5U is derived from a mouse. In other embodiments, A5U is derived from a rat. In some embodiments, A5U is derived from Drosophila.

[0053] In some embodiments, A5U is added to the cargo construct. Addition of A5U to the cargo construct allows for highly efficient cargo loading.

[0054] In some embodiments, the Arc 5'UTR comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:1. In some embodiments, the Arc 5'UTR comprises SEQ ID NO:1. In some embodiments, the Arc 5'UTR comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:2. In some embodiments, the Arc 5'UTR comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:3. In some embodiments, the Arc 5'UTR comprises SEQ ID NO:3. In some embodiments, the Arc 5'UTR comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the Arc 5'UTR comprises SEQ ID NO: 4.

[0055] Arc 3'UTR As used herein, the term "3'UTR sequence" refers to a 3' untranslated repeat sequence derived from an mRNA that can bind to a protein in an extracellular vesicle. For example, the Arc 3'UTR sequence can bind to the Arc protein in an extracellular vesicle. Such binding of 3'UTR to a protein can occur only by the 3'UTR sequence, or can occur when the 3'UTR sequence is linked to a non-Arc nucleic acid.

[0056] In some embodiments, the Arc mRNA does not include a 3'UTR sequence. In other embodiments, the Arc mRNA includes a 3'UTR sequence. In other embodiments, the Arc mRNA includes a 3'UTR sequence that is an Arc 3'UTR sequence. In some embodiments, the Arc mRNA includes a 3'UTR that is not an Arc 3'UTR sequence.

[0057] In some embodiments of the present disclosure, the Arc capsid gene is modified to allow for rapid clearance of Arc mRNA after translation into protein. In some embodiments, such modification is achieved by the addition of rat Arc 3'UTR sequence (partial A3U or full-length A3U) using various molecular cloning methods. Arc 3'UTR can be derived from human, mouse, rat (NCBI Gene ID: 23237, 11838, 54323), or Drosophila (dArc1; NCBI Gene ID: 36595). These embodiments provide a map of an exemplary DNA plasmid cloned to generate an exemplary capsid for engineered EV (Figure 17). Rapid removal of Arc mRNA after translation avoids overexpression of Arc in target cells without interfering with carrier production (Figure 18A). The present disclosure further includes the addition of the A3U sequence to sequences encoding full-length Arc proteins, Arc protein motifs, and any codon-optimized versions of these motifs from all species. The present disclosure includes both Arc mRNA sequences with and without the A3U sequence.

[0058] In some embodiments, the Arc 3'UTR is a mammalian Arc 3'UTR. In further embodiments, the Arc 3'UTR is a human Arc 3'UTR. In other embodiments, the Arc 3'UTR is derived from a mouse Arc 3'UTR. In other embodiments, the Arc 3'UTR is derived from a rat Arc 3'UTR. In some embodiments, the Arc 3'UTR is not a mammalian Arc 3'UTR. In further embodiments, the Arc 3'UTR is derived from a Drosophila Arc 3'UTR.

[0059] In some embodiments, the Arc 3'UTR comprises a nucleic acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:5. In some embodiments, the Arc 3'UTR mRNA comprises SEQ ID NO:5. In some embodiments, the Arc 3'UTR comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:6. In some embodiments, the Arc 3'UTR comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:7. In some embodiments, the 3'Arc UTR comprises SEQ ID NO:7. In some embodiments, the Arc 3'UTR comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 8. In some embodiments, the Arc 3'UTR comprises SEQ ID NO:8.

[0060] cargo In some embodiments, the compositions of the present disclosure (e.g., Arc EV) comprise a cargo. In some embodiments, the Arc EV comprises a cargo mRNA. As used herein, the term "cargo mRNA" refers to any nucleic acid that is not part of the Arc gene or is transcribed from the Arc gene. In some embodiments, the cargo mRNA encodes a therapeutic protein. In some embodiments, the cargo mRNA encodes a peptide, an enzyme, a cytokine, a hormone, a growth factor, an antigen, an antibody, a portion of an antibody, a clotting factor, a regulatory protein, a signaling protein, a transcription protein, and / or a receptor. In some embodiments, the cargo mRNA encodes a reporter protein. In some embodiments, the cargo mRNA encodes a fluorescent protein, a bioluminescent protein, and / or a recombinase reporter. In some embodiments, the cargo mRNA comprises a combination of a therapeutic protein and a reporter protein.

[0061] In some embodiments, cargo mRNA comprises Arc 5'UTR. In such an embodiment, cargo mRNA and Arc 5'UTR sequence are designed to be one continuous sequence, the sequence starts with the upstream Arc 5'UTR, followed by the cargo mRNA sequence portion that codes for the desired protein. In some embodiments, cargo mRNA is a chimeric mRNA, whose only 5'UTR is Arc 5'UTR, and whose coding portion is a non-Arc coding portion.

[0062] In some embodiments, the cargo mRNA does not include a 3'UTR sequence. In some embodiments, the cargo mRNA includes a 3'UTR sequence that is not an Arc 3'UTR sequence. In some embodiments, the cargo mRNA includes a 3'UTR sequence. In some embodiments, the cargo mRNA includes a 3'UTR sequence that is an Arc 3'UTR sequence.

[0063] In some embodiments, the nucleic acid molecule is an RNA polymer, such as a single-stranded RNA polymer, a double-stranded RNA polymer, or a hybrid of a single-stranded RNA polymer and a double-stranded RNA polymer. In some embodiments, the RNA comprises and / or codes for antisense oligoribonucleotides, siRNA, mRNA, tRNA, rRNA, snRNA, shRNA, microRNA, or non-coding RNA.

[0064] In some embodiments, the nucleic acid molecule comprises a hybrid of DNA and RNA.

[0065] In some embodiments, the nucleic acid molecule is an antisense oligonucleotide, optionally comprising DNA, RNA, or a hybrid of DNA and RNA.

[0066] In some embodiments, the nucleic acid molecule comprises and / or codes for an RNAi molecule.In some embodiments, the RNAi molecule is a microRNA (miRNA) molecule.In other embodiments, the RNAi molecule is a siRNA molecule.The miRNA and / or siRNA are optionally double-stranded or as hairpin miRNA and / or siRNA, and are also optionally packaged as precursor molecules.

[0067] In some embodiments, the nucleic acid molecule is a nucleic acid molecule for use in nucleic acid-based therapy.In some embodiments, the nucleic acid molecule is a nucleic acid molecule for regulating gene expression (for example, modulating the translation or degradation of mRNA), modulating RNA splicing, or RNA interference.In some cases, the nucleic acid molecule comprises and / or codes for antisense oligonucleotides, microRNA molecules, siRNA molecules, mRNA molecules, for use in regulating gene expression, modulating RNA splicing, or RNA interference.

[0068] In some embodiments, the nucleic acid molecule is a nucleic acid molecule for use in gene editing.Exemplary gene editing systems include but are not limited to CRISPR-Cas system, zinc finger nuclease (ZFN) system, and transcription activator-like effector nuclease (TALEN) system.In some embodiments, the nucleic acid molecule comprises and / or codes the components involved in CRISPR-Cas system, ZFN system, or TALEN system.

[0069] In some embodiments, the cargo is a therapeutic agent. In some embodiments, the cargo is a small molecule, a protein, a peptide, an antibody or a binding fragment thereof, a peptidomimetic, or a nucleotide mimetic. In some embodiments, the cargo is a therapeutic cargo, for example, including one or more drugs. In some embodiments, the cargo includes a diagnostic tool for profiling, for example, one or more markers (such as markers associated with one or more disease phenotypes). In further embodiments, the cargo includes an imaging tool.

[0070] In some embodiments, the nucleic acid molecule is a nucleic acid molecule for use in producing antigens for therapeutics and / or in producing prophylactic vaccines. For example, the nucleic acid molecule encodes an antigen that is expressed to elicit a desired immune response (e.g., a proinflammatory immune response, an anti-inflammatory immune response, a B cell response, an antibody response, a T cell response, a CD4+ T cell response, a CD8+ T cell response, a Th1 immune response, a Th2 immune response, a Th17 immune response, a Treg immune response, or a combination thereof).

[0071] In some embodiments, the nucleic acid molecule comprises a nucleic acid enzyme. A nucleic acid enzyme is an RNA molecule (e.g., a ribozyme) or a DNA molecule (e.g., a deoxyribozyme) that has catalytic activity. In some embodiments, the nucleic acid molecule is a ribozyme. In other embodiments, the nucleic acid molecule is a deoxyribozyme. In some cases, the nucleic acid molecule is an MNAzyme that functions as a biosensor and / or molecular switch (see, e.g., Mokany et al., JACS, 132(2):1051-1059 (2010)). Some embodiments of the present disclosure include RNA transcript compositions that include Arc mRNA as well as a cargo mRNA having an Arc 5'UTR sequence.

[0072] vector Some embodiments of the present disclosure include a recombination system comprising a first DNA encoding an Arc mRNA and a second DNA encoding a cargo mRNA having an Arc 5'UTR sequence. In some embodiments, the recombination system comprises a single construct comprising the first DNA and the second DNA. In some embodiments, the recombination system comprises a first construct comprising the first DNA and a second construct comprising the second DNA. In some embodiments, the construct further comprises a heterologous DNA regulatory element, where a "DNA regulatory element" is a DNA sequence that a certain transcription factor recognizes and binds to recruit or block RNA polymerase. In further embodiments, the heterologous DNA regulatory element comprises a promoter, an enhancer, a silencer, an insulator, or a combination thereof.

[0073] Each of the first and second nucleic acid sequences is operably inserted into an expression vector. In some embodiments, the first and second nucleic acid sequences are operably inserted into a common expression vector and expressed together. For example, in some embodiments, the second nucleic acid encoding the chimeric polynucleotide is inserted in frame into an intron of the first nucleic acid encoding the Arc protein. Methods for constructing expression vectors containing gene sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA methods, recombinant DNA synthesis methods, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning: Laboratory Manual (Cold Spring Harbor Press, Plainview, NY, 1989); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY, 1989).

[0074] Vectors include, but are not limited to, plasmids, viral nucleic acids, viruses, phage nucleic acids, phages, cosmids, and artificial chromosomes. Expression vectors generally contain regulatory sequences, which are elements necessary for the translation and / or transcription of the coding sequence inserted. For example, the coding sequence is preferably operably linked to a promoter and / or enhancer, which helps to control the expression of the desired gene product. The promoters used in biotechnology are of different types according to the type of control of gene expression that is intended. Promoters are generally divided into constitutive promoters, tissue-specific or developmental stage-specific promoters, inducible promoters, and synthetic promoters.

[0075] Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, In mammalian cell systems, promoters derived from mammalian genes or from mammalian viruses are preferred.

[0076] How to make Arc extracellular vesicles Some embodiments of the present disclosure include a method of producing EVs, comprising obtaining cells comprising Arc mRNA and a cargo mRNA comprising Arc 5'UTR; growing the cells in a medium under conditions that allow expression of the Arc protein encoded by the Arc mRNA, whereby the cells produce extracellular vesicles comprising the Arc protein and the cargo mRNA having the Arc 5'UTR sequence; and isolating the extracellular vesicles from the medium. In some embodiments, the method of producing EVs comprises obtaining cells comprising Arc mRNA and a cargo mRNA comprising Arc 5'UTR by introducing into a donor cell a DNA construct that is transcribed into Arc mRNA and a DNA construct that is transcribed into a cargo mRNA. In some embodiments, the method of producing EVs comprises obtaining cells comprising Arc mRNA and a cargo mRNA comprising Arc 5'UTR by introducing into a donor cell Arc mRNA and a cargo mRNA comprising Arc 5'UTR.

[0077] As used herein, "donor cell" is a technical term. A donor cell functions in that a construct is inserted into the donor cell and the cell produces EVs. For example, when a cargo mRNA containing Arc mRNA and Arc 5'UTR is inserted into a donor cell by methods known and described below, the donor cell translates Arc mRNA to synthesize Arc polypeptide. The Arc polypeptide then forms EVs through its retrovirus-like budding mechanism (which is exactly like the retrovirus-like budding mechanism of Gag). Since all cells produce EVs, all cells can be donor cells.

[0078] In some embodiments, EVs are created in prokaryotes, eukaryotes, or viruses. In some embodiments, engineered EVs are made in yeast, bacteria, viruses, protists, or other types of cells, whether they are unicellular organisms, multicellular organisms, or non-organisms that contain DNA.

[0079] Donor cells can provide EVs with their targeting specificity due to the natural ability of EVs derived from different donor cell types in targeting various tissues.

[0080] In some embodiments, the method of making EVs further comprises donor cells selected from neural cells, epithelial cells, endothelial cells, hematopoietic cells, connective tissue cells, muscle cells, bone cells, chondrocytes, germline cells, adipocytes, stem cells, autologous ex vivo differentiated cells, iPSC-derived ex vivo differentiated cells, cancer cells, and combinations thereof. In further embodiments, the donor cells are white blood cells. In some embodiments, the donor cells are autologous ex vivo differentiated white blood cells. In some embodiments, the donor cells are autologous ex vivo differentiated monocytes, macrophages, dendritic cells, or combinations thereof. In some embodiments, the donor cells are iPSC-derived ex vivo differentiated white blood cells. In some embodiments, the donor cells are iPSC-derived ex vivo differentiated monocytes, macrophages, dendritic cells, or combinations thereof.

[0081] Donor cells are created through the introduction of two nucleic acids into donor cells, namely Arc mRNA and cargo mRNA conjugated to A5U. This introduction is through methods known in the art, including but not limited to physical methods such as direct microinjection, biolistic particle delivery, electroporation, sonoporation, and laser-based optical transfection; and chemical methods such as calcium phosphate, cationic polymer, lipofection, FuGene, or dendrimer transfection. In some embodiments, nucleic acid is introduced into donor cells through polyethyleneimine (PEI) complexation, electroporation, cationic lipid complexation, lipid nanoparticle-mediated delivery, microinjection, or the use of adenovirus vectors. Cargo mRNA does not require conjugation to A5U, but RNA transduction into recipient cells is less efficient and less stable without incorporating A5U, as seen in FIG. 3A2.

[0082] Applicable Some aspects of the present disclosure include a method for delivering mRNA to a recipient cell.In some embodiments, the method includes obtaining the extracellular vesicles described herein and contacting the extracellular vesicles with a recipient cell, wherein the extracellular vesicles fuse with the cell, thereby delivering desired mRNA to the recipient target cell.In some embodiments, the method for delivering mRNA to a recipient cell is carried out in vitro.In some embodiments, the method for delivering mRNA to a recipient cell is carried out in vivo, as described below.

[0083] In some embodiments of the present disclosure, mRNA is delivered to recipient cells to treat a disease, produce a protein, induce cell death, inhibit cell death, alter cellular senescence, induce immune tolerance, modulate an existing immune response, modify intracellular activity, modify cell behavior, or a combination thereof.

[0084] The eaEVs of the present disclosure may be applied in a wide range of therapeutics. In some embodiments, the EVs are used for the treatment of cancer. In some embodiments, the EVs are used to prevent and / or treat viral infections. In some embodiments, the EVs are used in the treatment and / or prevention of allergies. In some embodiments, the EVs are used to treat tissue organization. In some embodiments, the EVs are used to treat inflammatory diseases. For example, EVs derived from peripheral immune cells may deliver drugs to diseased cells under inflammatory conditions across the blood-brain barrier without affecting healthy tissues. Such EVs may also deliver drugs, preferably to inflammatory microenvironments. In further embodiments, the EVs deliver therapeutic agents to inflammatory tumor microenvironments. In some embodiments, the target of such EVs includes virally infected tissues for the treatment of viral infections. In other embodiments, the EVs contain cargo mRNAs used for gene therapy.

[0085] Some aspects of the present disclosure include a method for treating a subject with extracellular vesicles. In some embodiments, the method for treating a subject with EVs includes obtaining EVs and administering them to a subject in need thereof. In some embodiments, the EVs are administered orally, rectally, intravenously, intramuscularly, subcutaneously, intrauterinely, intracerebrovascularly, or intraventricularly. In some embodiments, the EVs include mRNA and guide RNA of CRISPR-associated proteins adapted for treating diseases, including genetic disorders. In some embodiments, the extracellular vesicles are administered for treating neurodegenerative diseases, aging-related disorders, brain tumors, inflammatory conditions, and specifically deliver RNA across the blood-brain barrier to inflamed brain tissue without affecting healthy cells. In some embodiments, the EVs include mRNA corresponding to tumor-associated antigens, and the extracellular vesicles are delivered as a cancer vaccine for treating cancer, including melanoma, colon cancer, gastrointestinal cancer, genitourinary cancer, and hepatocellular carcinoma. In some embodiments, the EVs are delivered to prevent infection with infectious diseases, i.e., for vaccination. In some embodiments, the EVs are delivered for the treatment of autoimmune diseases. A non-limiting example of the treatment of autoimmune diseases is the delivery of interleukin 1 receptor antagonist (IL-1ra) or recombinant form of anakinra mRNA for the treatment of rheumatoid arthritis, an autoimmune disease in which IL-1 plays a key role.

[0086] Another aspect of the present disclosure is a method of delivering a construct of cargo linked to A5U in vivo to a donor cell to produce EVs in vivo using endogenous Arc protein. In some embodiments, the construct is delivered as DNA. In some embodiments, the construct is delivered as RNA. In some embodiments, the construct is delivered to the donor cell by lipid nanoparticles, exosomes, viruses, or another gene delivery method.

[0087] Working Example The following examples are presented to illustrate the present disclosure and are not intended to be limiting in any way. EXAMPLES

[0088] Engineering, production, and isolation of eaEVs for loading and delivery of mRNA Arc vesicles were engineered, produced, isolated, and characterized to validate their ability to deliver mRNA in vitro (Figure 1A). The two components of this carrier system are the mRNA cargo and the Arc protein capsid, which are introduced into virtually any donor cell type to produce enveloped eaEVs with different homing / targeting capabilities for diverse applications. The cargo construct was engineered for efficient mRNA encapsulation, and an A5U sequence was added upstream of the cargo mRNA sequence (Figure 1A).

[0089] The rat Arc capsid was used for characterization since it is distinct from endogenous Arc in human / mouse cell lines as well as in an in vivo mouse model. To produce engineered Arc EVs (eaEVs), mRNA encoding Arc capsid and cargo GFP mRNA was delivered into human embryonic kidney (HEK293) cells and mouse macrophage (RAW264.7) cells (Figure 1A). Expansion / encompassing titration / time-course experiments were performed to optimize EV production (Figures 7-11). Various transfection methods were investigated, including DNA / RNA electroporation, DNA transfection with PEI (Figures 7 and 10), and RNA transfection with liposomes (Figures 8-10). The dose of transfection reagent was carefully titrated to ensure that donor cell viability was not compromised after transfection (Figure 7, Figure 8), to ensure that the ratio of capsid / cargo constructs maximized mRNA encapsulation without introducing excessive amounts of capsid mRNA (Figure 9), and to ensure time course of mRNA loading and cargo expression in donor / recipient cells to maximize EV recovery efficiency (Figure 11). Finally, liposome-mediated RNA transfection was used to deliver 12 pmol of Arc (4.63 μg), 8 pmol of GFP (1.86 μg), and 8 pmol of A5U-GFP (2.26 μg) mRNA per million donor cells to produce control EVs and eaEVs over a period of 8–40 min in serum-free culture medium. The eaEV subpopulation isolated from the supernatant culture medium by ultrafiltration was labeled with fluorescent Arc antibody as characterized via fluorescent nanoparticle tracking analysis (NTA), while total EVs were measured by light scattering from all particles (Fig. 1B-1C and Fig. 12A-12C). A general EV marker antibody (anti-CD63) and a cell membrane dye (CellMask) were also used to label total EVs. The Arc+ EV subpopulation appeared to be larger than Arc vesicles (Fig. 1D and Fig. 12A'-12C'). The addition of rat Arc mRNA increased Arc+ EVs by 6.5-fold, suggesting efficient production of eaEVs (Fig. 1E-1F).

[0090] Next, eaEVs and Arc capsids were examined by negative staining electron microscopy (NSEM, Fig. 1G) to further characterize their size and shape. The fluorescence intensity of labeled eaEVs and total EVs was measured and correlated with the NTA results, and a correlation coefficient was calculated (Fig. 1H and Table S1). Together, the fluorescence intensity measurements were used to calculate the absolute particle concentration of eaEVs in total EVs using a fluorescence intensity reader. This confirmed the shape, size distribution, production efficiency, and sufficient purity of the recovered eaEVs.

[0091] To visualize the secretion and transfer of individual eaEVs, Arc capsid protein and cargo mRNA were labeled by confocal laser scanning microscopy (CLSM) followed by immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH) with quantitative hybridization chain reaction (qHCR) (Figure 1I). In addition to extracellular Arc+ / GFP+ eaEVs, their extracellular budding (possibly also endocytosis) was frequently observed, whereas release of cargo-containing Arc+ EVs from multivesicular bodies (MVBs) was a rare event (Figure 1I). EVs have been classified into ectosomes and exosomes based on differences in their size and biogenesis. Similar to its homolog, the viral capsid gag protein, Arc was thought to mediate direct extracellular budding of ectosomes, which are larger than exosomes released from MVBs fused with the donor cell membrane. Indeed, NTA size distribution confirmed imaging with donor cell cultures that eaEVs are more likely to be ectosomes than exosomes (Figure 1D and Figures 12A'-12C'). Upon transduction into recipient cells, successful uptake of eaEVs and translation of cargo in recipient RAW264.7 cells was demonstrated by live cell epifluorescence microscopy (EFM) for EV membrane staining and GFP expression (Figure 1J). GFP was observed only in recipient cells with CMDR (CellMask Deep Red), a cell membrane dye used to label EVs after their isolation from donor cell cultures and prior to their introduction into recipient cells. Taken together, this data demonstrates engineered, produced, and isolated eaEVs capable of loading and delivering mRNA. EXAMPLES

[0092] A5U-eaEVs can be loaded with mRNA with improved potency and selectivity Selective packaging of the 5'UTR of the HIV-1 genome depends on the Gag protein intact capsid (CA) domain lattice. Thus, the Arc protein can bind to A5U through ionic interactions at its N-terminus.

[0093] Thus, it was hypothesized that the addition of A5U would significantly improve the loading efficiency of mRNA cargo. Thus, to enable efficient cargo loading, rat A5U was added to the cargo construct because it shows greater similarity in the predicted secondary structure of the 5'UTR of the HIV1 RNA genome compared to A5U of other species (Figure 2A). A5U-GFP and GFP mRNA transcripts, cargo constructs with and without 5'UTR, were transcribed in vitro using fluorescently labeled Cy3-UTP and Cy5-UTP, respectively (NEB; CleanCap, T7-AG, with pseudoUTP). EVs were produced by transfecting these mRNA combinations into donor cells for six control and experimental groups: (1) mock transfection (NT); (2) Arc; (3) GFP; (4) A5U-GFP; (5) Arc / GFP; (6) Arc / A5U-GFP. Fluorescence intensity readings for isolated control and engineered EVs carrying cargo Cy3+ A5U-GFP and Cy5+ GFP were performed to compare the cargo loading efficiency of these EVs. The results showed that Arc significantly promoted the loading of mRNA into EVs (Figures 2B and 2E). Epifluorescence microscopy of donor cell cultures showed a significant increase in the amount of extracellular Cy3+ / Cy5+ cargo mRNA, confirming these results (Figures 2C-2D and 2F-2G).

[0094] To further characterize the cargo inside Arc capsids, RNA immunoprecipitation (RIP) followed by RT-qPCR was performed. After lysis of the EV outer membrane, Arc antibody was used to immunoprecipitate the Arc protein capsid, which was then digested to release its mRNA cargo for quantification by RT-qPCR. These experiments showed that A5U increases the encapsulation of cargo mRNA (Figure 2H). By optimizing the ratio of capsid to cargo construct transfected into donor cells, engineered Arc EVs allowed efficient (Figure 2I) and selective (Figure 2J) loading of A5U-GFP among numerous other RNAs, including rArc, GAPDH, cytosolic housekeeping genes, and 18S, one of the most prominent RNA species found in ectosomes.

[0095] Despite overexpression of capsid Arc mRNA in donor cells, the cargo A5U-GFP was preferentially enriched in capsids over Arc itself, unless excess Arc mRNA was transfected to compete for inclusion. When a ratio of (1xArc:3xA5U-GFP) was used, Arc mRNA was observed exclusively in any extracellular eaEVs (Figures 2K-2Q). Recipient HEK293 cells carrying A5U-eaEVs (Arc:A5U-GFP=1:3) were studied by immunocytochemistry (ICH) and fluorescent in situ hybridization (FISH) with quantitative hybridization chain reaction (qHCR), followed by confocal laser scanning microscopy (CLSM). Capsid proteins, cargo mRNA, and capsid mRNA in each individual EV were visualized at ultra-high resolution, and their overlap was quantified by ImageJ using a custom ImageJ macro code. This revealed that eaEV preferentially loaded cargo RNA with A5U compared to capsid Arc mRNA without A5U. On the other hand, when the ratio of (3×Arc:1×A5U-GFP) was introduced, significant amounts of Arc and 18S would be encapsulated in Arc capsids (Figure 2J). When the ratio of the transfection components capsid and cargo RNA was further optimized, it was found that when the same amount of cargo mRNA was present, GFP expression in donor cells decreased when more capsid mRNA was added (Figure 9). This was because more capsids encapsulating cargo mRNA were synthesized, reducing the amount of free mRNA left for translation. Considering all these factors, we decided to transfect a ratio of [1.5 × Arc (12 pmoles):1 × A5U-GFP (8 pmoles) per million cells] to achieve efficient and selective cargo loading without introducing excessive amounts of Arc mRNA.

[0096] Arc plays a vital role in the CNS and its overexpression in drug delivery systems should be avoided. For this reason, we optimized the ratio between transfection components through thorough characterization, both of which were driven by high-resolution qHCR and ultrasensitive RIP-qPCR, allowing single EV analysis with accurate quantification. Arc EVs were further engineered by adding A5U, an mRNA motif that allows highly efficient and selective packaging of mRNA cargo. EXAMPLES

[0097] A5U-eaEV may improve the efficacy and stability of delivery of mRNA cargo The efficacy and stability of A5U-eaEV as an mRNA drug carrier was verified by the increase in cargo mRNA uptake in recipient cells over a period of one week (Fig. 3A1). Interestingly, without A5U, transduction of recipient cells with RNA appeared to be less efficient and, more importantly, less stable (Fig. 3A2). The mechanism is likely similar to that of HIV gag, which requires its own genomic 5'UTR to stabilize the capsid. The behavior of EVs after their introduction into recipient cells is shown in Figures 3B1-3D6: after 15 min, EVs carrying fluorescent mRNA cargo began to dock to the recipient cell membrane (Figures 3B1-3B4); after 1 h, intracellular fluorescence was observed, and by 4 h, virtually all recipient cells had received Cy3+ A5U-eaEVs, whereas the control was less efficient (Figures 3C1-3C4); this advantage in uptake efficiency was greatly magnified over the time course, where not only did all cells have the cargo, but also the cargo in each cell increased (Figures 3A1 and 3D1-3D6). It is necessary to emphasize that the same amount of total EVs was added to each sample group. A rapid fluorescent readout was used as done in Example 1 above, using the dye CMDR to quantify total EVs. With careful optimization of the dye concentration, this method accurately quantified the relative amount of total EVs (Figure 3E and Figure 13). CMDR+ EVs were added to recipient cells and the mean fluorescence was measured 1 h later, and the results suggested that a similar number of total EVs were taken up by recipient cells (Figure 3F). Thus, A5U-eaEVs significantly improved the delivery efficiency and stability of mRNA cargo.

[0098] A consistent increase in GFP expression (Figure 3G) via A5U-eaEV delivery was also observed. Despite Arc / A5U-GFP substantially promoting mRNA encapsulation in donor cells and mRNA uptake by recipient cells, RAW264.7 recipient cells showed weak and sparse GFP expression. This is likely because the release and translation of eaEV cargo is dependent on activity not only in neurons but also in immune cells. In addition to RAW264.7, this increase in cargo translation was also verified in triple-negative breast cancer cells (MDA-MB-231; Figure 3H). In conclusion, A5U-eaEV can deliver mRNA with improved efficiency and stability in vitro. EXAMPLES

[0099] Leukocyte-derived A5U-eaEVs can efficiently deliver mRNA across the BBB and specifically target neuroinflammation The blood-brain barrier (BBB) ​​is a highly dynamic and selective semi-permeable boundary that separates the peripheral circulation from the central nervous system (CNS) and prevents the entry of macromolecular drugs into the brain. The BBB is composed of a chain of brain microvascular endothelial cells (BMECs), their tight junctions, basement membranes, pericytes, and astrocytes at the ends. BMECs normally express lower levels of leukocyte adhesion molecules compared to peripheral endothelial cells to prevent immune cell margination and migration into the brain. The BBB is disrupted by more severe pathological changes, such as age-associated low-grade inflammation, also referred to as inflammatory aging, neurodegenerative diseases, as well as systemic inflammation and secondary injuries (e.g., stroke). In response to these inflammatory stimuli derived from the brain, BMECs have been shown to exhibit increased permeability and leukocyte adhesion molecule expression, allowing more leukocytes (e.g., macrophages and DCs) and leukocyte-derived EVs to cross the BBB and enter the brain. Although leukocyte EVs enter the brain independently, without the involvement of brain-infiltrating immune cells, the accumulation of such EVs becomes increased in the inflamed brain, further increasing the permeability of the BBB, making EVs important candidates for drug delivery to the brain.

[0100] In addition to the neuroinflammation targeting ability of leukocyte EVs, the natural role of Arc EVs in interneuronal mRNA transfer further improves the uptake of these vesicles by neurons. Furthermore, the Arc capsid protects cargo mRNAs from degradation by RNases, increasing their stability until release is triggered. Given these natural advantages of leukocyte eaEVs and the improvements observed above that increase the loading of mRNA cargo, these EVs may be sufficient to deliver mRNA to the CNS and target neuroinflammation.

[0101] To generate a pool of immunologically inert EVs for in vivo studies, autologous donor leukocytes were differentiated ex vivo from BM cells harvested from mice with allogeneic major histocompatibility complex (MHC) haplotypes. Once isolated from the femur, BM cells were cultured with GM-CSF and IL-4 for 7 days (Figures 4A-4B). Having established that EVs derived from both macrophages and DCs cross the BBB, we utilized culture protocols for both populations and cultured subpopulations including, but not limited to, monocyte-derived DCs, monocyte-derived macrophages, and conventional DCs (Figure 4C).

[0102] As described above, control and experimental EVs were produced, isolated, and characterized by transfecting with (1) mock-transfected negative control (NC); (2) Arc; (3) GFP; (4) A5U-GFP; (5) Arc / GFP; and (6) Arc / A5U-GFP. BM-DCs / macrophages could internalize their own EVs (Figure 4D). Fine-tuning the balance between secretion and internalization was crucial. For these experiments, EVs were produced for 24–48 min depending on donor cell confluency to reach saturation of EVs in the supernatant medium, and total EV concentration was monitored and measured via CMDR epifluorescence intensity in a time-course experiment. Before harvesting, each control and experimental group was verified to contain approximately the same amount of total EVs. Despite the difference in the ratio of eaEVs, the Arc / A5U-GFP group showed the largest ratio of eaEVs among all EVs (Figure 4F). This difference may be due to higher production or stability by A5U-eaEVs, since this measurement was made 42 hours after transfection (40 hours production + 2 hours purification / staining), leaving enough time for EV degradation.To explore the potential of leukocyte eaEVs in targeting highly inflamed brain regions across the BBB, we intravenously (IV) injected equal amounts of total EVs per gram of mouse body weight into various neuroinflammation models.

[0103] To study the in vivo biodistribution of eaEVs in a pan-neuronal inflammation model, leukocyte EVs (9 × 10 per gram body weight) were 7eaEVs (total EVs) were injected intravenously (IV) into old (90 weeks old, weighing approximately 40 g) mice, and organs were harvested 72 hours after transcardial perfusion. Cy3+ / Cy5+ fluorescently labeled mRNA allowed visualization of eaEV biodistribution and cargo uptake via IVIS (in vivo imaging system). For IVIS analysis of total EV biodistribution, non-fluorescent mRNA was transfected and total EVs labeled with CMDR cell membrane dye were harvested. Various organs of these old mice (brain, liver, spleen, kidney, heart, lung) were perfused, fixed, sectioned, and imaged by IVIS. Similar levels of CMDR signal were observed between experimental and control groups, representing the biodistribution of total or degraded EVs in each organ at the time of harvest (3 days after IV injection) (Figure 14). However, Arc clearly increased mRNA delivery in vivo (Figure 4G-4H). Surprisingly, A5U further increased the accumulation of mRNA in the aged brain (Figures 4G and 4I), while GFP mRNA was also delivered by Arc EVs, mostly to the liver and kidney (Figures 4H and 4J). This increased mRNA uptake into the aged brain was similar to the in vitro uptake results (Figures 3B1-3B2), potentially resulting from increased eaEV production or higher stability. The formation of Arc capsids is known to require RNA. In mouse and human donor cells, the addition of rat A5U further stabilized rat Arc capsids, resulting in higher production efficiency, while at the same time stabilizing eaEVs. Thus, BM-DC / macrophage-derived A5U-eaEVs can specifically deliver mRNA across the BBB to target chronic pan-neuronal inflammation. EXAMPLES

[0104] Leukocyte-derived A5U-eaEVs can efficiently deliver mRNA across the BBB for pan-neuronal expression under chronic inflammation Protein translation from cargo mRNA was examined in the brain. A5U-eaEVs were administered systemically to deliver A5U-GFP mRNA to old mice (approximately 90 weeks old) and control young mice (<24 weeks old), and brains were harvested 2 and 6 days after administration. Neurons were labeled by immunohistochemistry (IHC) staining with NeuN (Fox-3; hexaribonucleotide-binding protein 3). High levels of neuronal GFP expression (NeuN+ / GFP+) were observed in aged brains compared to young controls (Figures 5A-5E). Interestingly, in young control brains, GFP was expressed in blood cells within microvasculature (Figure 5A', green, which is an inset of Figure 5A) or in infiltrating immune cells (Figure 5C, green), comparable to aged brains. However, only aged brains showed significant intraneuronal expression (Figure 5B', which is an inset of Figure 5B; Figure 5D, white). Certain brain regions (e.g., hypothalamus, Figures 5C-5D) took up and expressed more cargo than other regions (e.g., cerebral cortex). Collectively, this aging model supports the pan-neuronal delivery of mRNA by eaEVs throughout the whole brain in response to chronic inflammation. EXAMPLES

[0105] Leukocyte-derived A5U-eaEVs can efficiently deliver mRNA across the BBB for specific local expression under acute injury To study the potential of eaEVs to target focal inflammatory sites, we created a photothrombotic stroke model by inducing ischemic injury in a small area of ​​the mouse cerebral cortex via photoactivation of a photosensitive Rose Bengal dye injected intraperitoneally (Fig. 6A). Twenty-four hours after stroke induction, leukocyte EVs (3 × 10 per gram body weight) were injected into the brain. 7Total EVs (1000 total EVs) were injected IV and brains were harvested 2 days after EV administration. High level GFP expression in NeuN+ neurons in the traumatized brain area marked by increased Iba1 (a microglia / macrophage marker to assess the level of inflammation, Fig. 6B) suggested that leukocyte-derived eaEVs delivered mRNA across the BBB and concentrated at the site of inflammation upon injury (Fig. 6C-6G). Within the traumatized area, neurons were damaged and reduced the number of NeuN+ cells (Fig. 6H). The site was highly inflammatory as the count of Iba1+ cells increased, attracting microglia and infiltrating macrophages (Fig. 6H). The number of GFP+ cells increased (Fig. 6H). Thus, leukocyte eaEVs may deliver mRNA across the BBB and locally target injury-induced inflammation without affecting healthy cells in the same brain.

[0106] In conclusion, a safe and effective drug carrier for mRNA with high cargo loading and delivery efficiency was engineered. The method of producing and isolating eaEVs was optimized, leading to detailed profiling of cargo loading and transduction efficacy. Furthermore, the ability of eaEVs to cross the BBB and deliver mRNA to animal models of neuroinflammation was demonstrated, indicating their potential use in novel therapeutics for inflammatory conditions in the CNS. EXAMPLES

[0107] Leukocyte-derived A5U-eaEVs can deliver mRNA that penetrates deep into solid tumors In addition to neuroinflammation, eaEVs were investigated in the tumor inflammatory microenvironment. Chronic inflammation and increased permeability are also hallmarks of cancer, and both exogenous and endogenous factors, such as immune regulatory imbalance, carcinogen exposure, and genetic alterations resulting in oncogene activation or loss of tumor suppressors, can induce inflammatory responses within the tumor microenvironment (TME). TME vasculature often possesses abnormal morphology associated with leaky, disorganized, immature, thin-walled, and poorly perfused vascular networks caused by poor pericyte coverage and disruption of the supporting basement membrane of endothelial cells. Thus, it was hypothesized that leukocyte-derived eaEVs could deliver mRNA to the TME.

[0108] First, MDAMB231 cells (3 × 10 6 A triple-negative breast cancer (TNBC) mouse model was generated via subcutaneous injection of 1000 ng / mL of the IgG1 gene into mice (4-6 week-old female NIH-III nude mice). The tumor volume was approximately 1,000 mm. 3Once the mice reached 100 days of age (approximately 2 weeks of age), control and eaEVs carrying GFP cargo mRNA or A5U-GFP cargo mRNA were injected IV. EVs were produced, isolated, characterized, and quantified as described above, and the same amount of total EVs was injected per gram of mouse body weight. To study the in vivo biodistribution of eaEVs within the TME, organs were harvested 3 days after IV injection for IVIS analysis. Before excising the organs, transcardial perfusion was performed to remove residual eaEVs in the circulation. Here, non-fluorescent mRNA and total EVs labeled with CMDR membrane dye were transfected. A significant increase in CMDR signal was observed in the tumor with Arc+ EVs, whereas other organs showed similar CMDR levels (Figure 15A). In this experiment, the distribution of CMDR+ EVs was not discernible from that of the dye itself. Animals receiving leukocyte eaEVs showed comparable amounts of CMDRs in tumors as in liver and kidney, both of which are involved in lipid metabolism (Figure 15B). Despite having ample CMDR accumulation, significantly higher levels of GFP were expressed in tumors as shown by confocal imaging at subcellular resolution (Figure 15C). Translation of cargo mRNA was then examined in tumors that were excised, fixed, thickly sectioned, washed, and IHC stained for K-Ras prior to CLSM. The results suggest that eaEVs penetrated deep into tumor tissue to express mRNA cargo, whereas control EVs were distributed only in the vicinity of the pancreatic duct (Figures 15D-15E). Taken together, these results confirmed that deep tumor penetration of mRNA-loaded eaEVs allows efficient uptake and translation of mRNA.

[0109] Since the addition of Arc improves tumor targeting, antitumor small molecule drugs were loaded into eaEVs to explore the potential of eaEVs in antitumor therapy. These drugs were loaded using three methods: (1) adding small molecule drugs to the culture medium of donor cells after DNA / RNA transfection of capsid and cargo constructs; (2) incubating small molecule drugs with EVs purified from donor cell cultures; and (3) loading small molecule drugs into purified eaEVs by low-power sonication (6 cycles of 30 s on / off for a total of 3 min with 2 min cooling). The small molecule drugs were successfully loaded and delivered to recipient triple-negative breast cancer cells (Figure 16). Thus, the tumor targeting feature of eaEVs can be used to deliver small molecule drugs deep into tumors.

[0110] In conclusion, a safe and effective drug carrier for mRNA with high cargo loading and delivery efficiency was engineered. The methods for producing and isolating eaEVs were optimized, leading to detailed profiling of cargo loading and transduction efficacy. Furthermore, eaEVs enabled deep tumor penetration and efficient mRNA delivery in an animal model of breast cancer, supporting their potential in novel cancer therapy.

[0111] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

Claims

1. A RNA transcript composition, comprising a cargo mRNA comprising an Arc 5'UTR sequence and an Arc mRNA.

2. The Arc 5'UTR sequence includes an Arc 5'UTR sequence derived from a mammal, such as a human, a mouse, or a rat, or derived from Drosophila; 2. The composition of claim 1, optionally wherein the Arc 5'UTR sequence comprises a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 1-4.

3. The composition of claim 1 or 2, wherein the cargo mRNA further comprises a poly(A) signal.

4. The cargo mRNA is A therapeutic protein; or a peptide, an enzyme, a cytokine, a hormone, a growth factor, an antigen, an antibody, a portion of an antibody, a clotting factor, a regulatory protein, a signal transduction protein, a transcription protein, and / or a receptor; or Fluorescent proteins, bioluminescent proteins, and / or recombinase reporters The composition according to any one of claims 1 to 3, which encodes:

5. Arc mRNA comprises the Arc 3'UTR sequence, Optionally, the Arc 3′UTR sequence is derived from a mammal, such as a human, mouse, or rat, or from Drosophila; and / or 5. The composition of any one of claims 1 to 4, optionally wherein the Arc 3'UTR sequence comprises a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 5 to 8.

6. The composition of any one of claims 1 to 5, wherein the Arc mRNA further comprises a poly(A) signal.

7. The composition of any one of claims 1 to 6, wherein the Arc mRNA encodes an Arc protein derived from a mammal, such as human, mouse, or rat, or derived from Drosophila.

8. The Arc mRNA comprises an Arc mRNA sequence derived from a mammal, such as a human, mouse, or rat, or derived from Drosophila; Optionally, the Arc mRNA comprises a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs:9-12.

9. A recombinant system comprising a DNA encoding a cargo mRNA comprising an Arc 5'UTR sequence, and a second DNA encoding an Arc mRNA.

10. The system according to claim 9, wherein the cargo mRNA is as defined in any one of claims 2 to 8.

11. The system comprises a single plasmid comprising a DNA encoding a cargo mRNA having an Arc 5'UTR sequence and a second DNA encoding an Arc mRNA; or A first plasmid containing a DNA encoding a cargo mRNA having an Arc 5'UTR sequence and a second plasmid containing a second DNA encoding an Arc mRNA. The system according to claim 9 or 10, comprising:

12. The plasmid(s) further comprises a heterologous DNA regulatory element, The system of claim 11 , wherein the heterologous DNA regulatory element optionally comprises a promoter, enhancer, silencer, insulator, or a combination thereof.

13. An extracellular vesicle comprising: Arc protein; and Cargo mRNA containing the Arc 5'UTR sequence Including, Optionally, further comprising one or more small molecule drugs; Vesicle.

14. The cargo mRNA is as defined in any one of claims 2 to 4, Arc protein is Optionally as defined in claim 7; and / or Optionally, comprising at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOs: 13-16; 14. The vesicle of claim 13.

15. 1. A method for producing extracellular vesicles, comprising: (a) Cells containing Arc mRNA and a cargo mRNA containing the Arc 5′UTR. obtaining (b) growing the cells in a medium under conditions that allow expression of an Arc protein encoded by the Arc mRNA, wherein the cells produce extracellular vesicles that contain the Arc protein and a cargo mRNA having an Arc 5'UTR sequence; and (c) Separating the extracellular vesicles from the medium The method includes:

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