Modified small extracellular vesicles and uses thereof

Abstract

Small extracellular vesicles (sEVs) are provided herein containing at least one tetraspanin that has at least one noncanonical amino acid (ncAA) substituting for a native amino acid in an extra-exosomal loop. The tetraspanin can be CD81, and the ncAA can contain an azide group, a carboxyl group, or a strained alkene. The sEV can further include a peptide that decreases the elimination of sEV by macrophages, a peptide that steers the sEV to a target tissue, and / or a peptide that decreases the rate of degradation of the sEV by target cell lysosomes, each attached to a ncAA. The sEV can further include a poly-histidine tag and / or cargo. The associated compositions, plasmids, cell lines, and animals, methods of producing the sEVs, and methods of treating a disease using the sEV or sEV compositions are also provided.

Description

MODIFIED SMALL EXTRACELLULAR VESICLES AND USES THEREOFInventors: Janos ZempleniJiantao GuoGOVERNMENT SUPPORT[00011 This invention was made with government support under P20 GM104320 and P30 GM154608 awarded by the National Institutes of Health, and 2022-67021-36407 awarded by the National Institute of Food and Agriculture. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 547,981 filed on November 9, 2023, the content of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0003] The disclosure relates to small extracellular vesicles and other delivery compositions that contain a tetraspanin with at least one noncanonical amino acid each substituting for a native amino acid in an extra-exosomal loop.BACKGROUND

[0004] Synthetic and cell culture-derived nanoparticles (small extracellular vesicles, sEVs, “exosomes”) have attracted considerable attention and yielded successes in the prevention, treatment, and cure of disease by delivering therapeutics to sites of disease. sEVs are secreted by virtually every' living cell and are detectable in all body fluids studied to date. sEVs facilitate intercellular communication by transferring endogenous regulatory cargos (proteins, lipids, RNA) from donor cells to recipient cells. Milk sEVs ("MEs"). such as from bovine milk, ovine milk, or caprine milk, are a particular type of sEVs . However, the following challenges currently limit the use of MEs (and any other nanoparticle) in the delivery of therapeutics: 1) MEs (and therapeutic cargo) are rapidly eliminated by circulating and tissue macrophages; 2) MEs (and therapeutic cargo) do not accumulate specifically in a diseased tissue or cell ty pe that is the target of treatment; and 3) a large fraction of MEs (and therapeutic cargo) may be degraded in lysosomes upon internalization by target cells.SUMMARY[0005| Provided herein are small extracellular vesicle (sEV) compositions containing at least one tetraspanin that has at least one noncanonical amino acid (ncAA) substituting for a native amino acid in an extra-exosomal loop. The tetraspanin can be CD81 , and the ncAA can include those with various functional groups, such as: an azide group, exemplified by p-azido-L- phenylalanine; a carbonyl group, as in 2-amino-8-oxononanoic acid; and strained alkenes, represented by trans-cyclooct-2-en-L-lysine (such as N6-(3-(cyclobut-2-en-l- yl)propanoyl)lysine). The sEV can further include a peptide that decreases the elimination of sEV by macrophages, a peptide that steers the sEV to a target tissue, and / or a peptide that decreases the rate of degradation of the sEV by target cell lysosomes, each attached to a ncAA. The sEV can further include a poly -histidine tag and / or be loaded with cargo. The associated compositions, plasmids, cell lines, and animals, methods of producing the sEVs, and methods of treating a disease using the sEV or sEV composition are also provided.[0006 In one aspect, provided is a small extracellular vesicle (sEV) containing at least one tetraspanin, the at least one tetraspanin containing at least one noncanonical amino acid (ncAA) substituting for a native amino acid in an extra-exosomal loop. The sEV can be derived from milk, such as bovine milk, ovine milk, or caprine milk. The EVs can be derived from cells (such as a cell line), and can be of a size, morphology7, and bioavailability7identical to an sEV derived from bovine milk or non-bovine sources, such as ovine or caprine milk. The tetraspanin containing ncAA can be selected from the group consisting of CD81, CD9. and CD63. In specific embodiments, the tetraspanin containing ncAA is CD81 . In specific embodiments, the tetraspanin containing ncAA is CD63. In some embodiments, one tetraspanin contains three ncAAs substituting for three native amino acids.The ncAAs can include those with various functional groups, such as: an azide group, exemplified by p-azido-L-phenylalanine; a carbonyl group, as in 2-amino-8-oxononanoic acid; and strained alkenes, represented by trans-cyclooct-2-en-L-lysine. In specific embodiments, the ncAA is p-azido-L-phenylalanine, 2-amino-8-oxononanoic acid, and N6-(3-(cyclobut-2-en- l-yl)propanoyl)lysine.[0007| In the sEVs, a peptide that decreases the elimination of sEV by macrophages can be attached to a first ncAA, a peptide that steers the sEV to a target tissue can be attached to a second ncAA, and / or a peptide that decreases the rate of degradation of the sEV by target cell lysosomes can be attached to a third ncAA. As examples and without limitation, the peptide that decreases the elimination of sEV by macrophages can include CD24 or CD47, the peptide that steers the EV to the target tissue can include apoE or CooP, and / or the peptide thatdecreases the rate of degradation of the sEV by target cell lysosomes can include poly-arginine or Alix. The sEV can further contain a poly -histidine tag in the tetraspanin.[«O08| In some embodiments, the sEV is loaded with cargo. The cargo can contain a gene editing reagent, a protein, a plasmid, and / or a small interfering RNA. The gene editing reagent can include a Cas9 enzy me and a guide RNA. The plasmid can encode Syngapl and / or Magel2. [000 [ The sEV can further contain a detectable label. The detectable label can contain a fluorophore and / or a pH-sensitive dye.

[0010] In one aspect, provided is a cell line capable of producing the sEV provided herein.[00111 The cell line can be a mammary cell line, such as a mammary alveolar MAC-T cell line or a mammary epithelial HME1 cell line. The genome of the cell line can contain at least one amber mutation site encoding the at least one ncAA and a poly -histidine tag, to be expressed in a loop on the surface of the at least one tetraspanin, a biosynthetic enzyme that converts at least one ncAA precursor to the at least one ncAA, and / or at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin. For example, the genome of the cell line can contain three amber or quadruplet codon mutation sites or combinations thereof, each encoding an ncAA and a poly -histidine tag, to be expressed in a loop on the surface of CD81, and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent CD81. In certain embodiments, the genome of the cell line can contain three amber and quadruplet codon mutation sites, each encoding an ncAA and a poly -histidine tag, to be expressed in a loop on the surface of CD81.[0012J In one aspect, provided herein is a plasmid containing a nucleic acid sequence encoding: at least one tetraspanin containing at least one amber mutation site encoding at least one noncanonical amino acid (ncAA) and a poly -histidine tag, to be expressed in a loop on the surface of the at least one tetraspanin. a biosynthetic enzyme that converts at least one ncAA precursor to the at least one ncAA, and / or at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin. In specific embodiments, the plasmid contains a nucleic acid sequence encoding: CD81 containing three amber and quadruplet codon mutation sites each encoding an ncAA and a poly -histidine tag (for example His6 tag), to be expressed in a loop on the surface of the at least one tetraspanin, and / or three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent tetraspanin.|0013] In one aspect, provided is a transgenic animal capable of producing the sEV provided herein. In some embodiments, the transgenic animal is a cow, and the milk of the cow contains the sEV provided herein. A genome of the transgenic animal can contain one or more of anamber mutation site encoding the at least one ncAA and a poly -histidine tag to be expressed in a loop on the surface of the at least one tetraspanin, a biosynthetic enzyme that converts at least one ncAA precursor to the at least one ncAA, and / or at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin. In some embodiments, the genome of the transgenic animal contains three amber or quadruplet codon mutation sites or combinations thereof, each encoding an ncAA and a poly -histidine tag to be expressed in a loop on the surface of CD81, and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent CD81. In certain embodiments, the genome of the transgenic animal contains three amber and quadruplet codon mutation sites, each encoding an ncAA and a poly -histidine tag, to be expressed in a loop on the surface of CD81. |0014] In one aspect, provided is a pharmaceutical composition containing a therapeutically effective amount of the sEV provided herein.

[0015] In one aspect, provided is a method of treating a subject having a disease. The method includes administering a therapeutically effective amount of the sEV or the pharmaceutical composition provided herein. In some embodiments, a peptide that steers the sEV to a diseased tissue is attached to the at least one ncAA. In some embodiments, the disease is glioblastoma multiforme, and the peptide that steers the sEV to the diseased tissue contains apoE or CooP. In some embodiments, the disease is Syngapl haploinsufficiency, and the sEV contains a plasmid encoding Syngapl. In some embodiments, the disease is Magel2 associated disease, and the sEV contains a plasmid encoding Magel2. In some embodiments, the disease is selected from the group consisting of Alzheimer’s disease, pancreatic cancer, heart disease, cystic fibrosis, Huntington’s disease, and Duchenne muscular dystrophy.[0016| In one aspect, provided is a method of producing a modified small extracellular vesicle (sEV). The method includes: introducing into a genome of a cell line at least one amber mutation site encoding the at least one ncAA, and a poly -histidine tag to be expressed in a loop on the surface of the at least one tetraspanin, and at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin; culturing the modified cell line in a cell culture medium; and isolating the sEV from the cell culture medium. In some embodiments, the cell line is a mammary alveolar MAC-T cell line or a mammary epithelial HME1 cell line.

[0017] In one aspect, provided is method of producing a modified small extracellular vesicle (sEV). The method includes: introducing into a genome of an animal at least one amber mutation site encoding the at least one ncAA, and a poly -histidine tag to be expressed in a loop on the surface of the at least one tetraspanin, and at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin; collecting milk produced by the animal; and isolating the sEV from the milk. The animal can be a ruminant, such as a cow.|0018] In some embodiments, the methods include introducing into the genome three amber or quadruplet codon mutation sites or combinations thereof, each encoding an ncAA, and a poly -histidine tag, to be expressed in a loop on the surface of CD81, and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent CD81. In certain embodiments, the methods include introducing into the genome three amber and quadruplet codon mutation sites, each encoding an ncAA, and a poly-histidine tag, to be expressed in a loop on the surface of CD81.|(IO19] The ncAAs can include those with various functional groups, such as: an azide group, exemplified by p-azido-L-phenylalanine; a carbonyl group, as in 2-amino-8-oxononanoic acid; and strained alkenes, represented by trans-cyclooct-2-en-L-lysine. In some embodiments, the three ncAAs include one or more of p-azido-L-phenylalanine, 2-amino-8-oxononanoic acid, and N6-(3-(cyclobut-2-en-l-yl)propanoyl)lysine.[0020| In some embodiments, the method further includes attaching a peptide that decreases the elimination of sEV by macrophages to a first ncAA, attaching a peptide that steers the sEV to a target tissue to a second ncAA, and / or attaching a peptide that decreases the rate of degradation of the sEV by target cell lysosomes to a third ncAA. The peptide that decreases the elimination of sEV by macrophages can include CD24 or CD47, the peptide that steers the EV to the target tissue can include apoE or CooP, and / or the peptide that decreases the rate of degradation of the sEV by target cell lysosomes can include poly-arginine or Alix.BRIEF DESCRIPTION OF THE DRAWINGS[00211 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.

[0022] The accompanying drawings, which are included to provide a further understanding of the embodiments of the present disclosure, are incorporated in and constitute a part of this specification, illustrate embodiments of the present disclosure, and together with the detailed description, serve to explain principles of the embodiments discussed herein. No attempt is made to show structural details of this disclosure in more detail than may be necessary for a fundamental understanding of the embodiments discussed herein and the numerous ways in which they may be practiced. According to customary practice, the various features of thedrawings discussed below are not necessarily drawn to scale. Dimensions of various features and elements in the drawings may be expanded or reduced to illustrate embodiments of the disclosure more clearly.|OO23] FIG. 1A schematically depicts small extracellular vesicles (sEVs) derived from milk (MEV) and its genetic modification. “hTERT-HMEE' is a human mammary' epithelial cell line HME1 immortalized using hTERT. The histidine tag (marked as “His6” in FIG. 1A) can be enzymatically removed after purification.[00241 FIG. IB schematically depicts sEV uptake, degradation, and retrofusion.

[0025] FIG. 2 schematically depicts three amber and quadruplet codon mutation sites and one poly -histidine (poly-his) tag to be inserted in the MAC-T cell genome.|0026] FIG. 3A schematically depicts incorporation of ncAA in proteins for subsequent attaching of synthetic peptides by bioorthogonal reaction.

[0027] FIG. 3B schematically depicts use of bioorthogonal (click) chemistry7to insert a ncAA to a tetraspanin (such as CD63) for expression in cells.[0028} FIG. 3C depicts expression of a CD63-iRFP fusion protein containing azido-Phe in iRFP.

[0029] FIG. 4A depicts exemplary size and count of sEVs by nanoparticle tracking analysis (NTA).

[0030] FIG. 4B depicts morphology and dispersion of sEVs by transmission electron microscopy (TEM).

[0031] FIG. 4C depicts positive and negative sEV markers. Red boxes indicate positive markers. apoB, apolipoprotein B-100; P-int., Butyr, butyrophilin; H3, histone H3; P-integrin; M, marker (protein ladder).

[0032] FIG. 5A schematically depicts a CD63-iRFP fusion protein.|0033] FIG. 5B schematically depicts MAC-T cells expressing a fusion protein containing an amber mutation. Translation of the fusion protein in the absence (top) and presence of ncAA (bottom).

[0034] FIG. 5C depicts MAC-T cells expressing a fusion protein containing an amber mutation. Cells (brightfield, top), iRFP fluorescence (middle), and merged images (bottom).

[0035] FIG. 6A schematically depicts a plasmid with a CMV promoter and iRFP coding sequence.|0036] FIG. 6B depicts expression of an iRFP plasmid, delivered by MEVs, in murine brains.

[0037] FIG. 7A and FIG. 7B depict MEVs labeled with a pH-sensitive dye.

[0038] FIG. 7C depicts MEV labeled with a pH-sensitive dye localizing to lysosomes Cl,pHrodo-labeled MEVs; C2, unlabeled MEVs; C3 nuclei stained with Hoechst Blue; C4, merged Cl, C2 and C4. N = 3 biological repeats.

[0039] FIG. 8A depicts expression of CD24 and CD47 mRNA in non-transformed (negative, neg.) and transformed (positive, pos.) MAC-T cells, n.d., not detectable.

[0040] FIG. 8B depicts the uptake of CD24 / CD47-negative, CD24-positive, and CD47- positive MEVs by BMDMs at t=2h (***P < 0.001, N = 3).[00411 FIG. 8C depicts accumulation of CD47-positive MEVs in murine brains. Brains were harvested 8 hours after nasal administration of MEVs (4.1 x 107 / g body wt ). CD47-negative MEVs and PBS served as wild-type and vehicle controls.

[0042] FIG. 9 depicts uptake of wild-type and apoE- and CooP -modified MAC-T BMEs in human IDH1(R132H) glioma cells. N = 3; *P < 0.05.

[0043] FIG. 10 depicts localization of modified MEVs to GBM tumors.

[0044] FIG. 11A depicts expression of Syngapl protein in SyngapT1' mice and WT mice treated with Syngapl plasmid-loaded MEVs or empty MEVs.[0045} FIG. 11B depicts the expression of Syngapl mRNA in the brain of SyngapT ~ mice and WT mice treated with Syngapl plasmid-loaded MEVs or empty MEVs.

[0046] FIG. 12 depicts effect of freezing on the MEV count in preparations of purified MEVs (*P<0.05 vs. fresh milk; N=3-4).

[0047] FIG. 13 depicts preservation of lyophilized MEs by addition of trehalose (N=l).DETAILED DESCRIPTION

[0048] The description may use the phrases “in certain embodiments,” “in various embodiments,” “in an embodiment,” or “in embodiments,” which may each refer to one or more of the same or different embodiments. Furthermore, the terms “comprising,” “including,” “having,” and the like, as used with respect to embodiments of the present disclosure, are synonymous. The term “about” or “approximately” are defined as being close to as understood by one of ordinary skill in the art. In one non-limiting embodiment, the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. The terms “removing,” “removed,” “reducing,” “reduced,” or any variation thereof, when used in the claims and / or the specification includes any measurable decrease of one or more components in a mixture to achieve a desired result. The use of the words “a” or “an” when used in conjunction with any of the terms “comprising,” “including,” “containing,” or “having,” in the claims or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The term “plurality” asused herein refers to two or more items or components. The terms “wt.%,’" “vol.%,” or “mol.%” refers to a weight, volume, or molar percentage of a component, respectively, based on the total weight, the total volume of material, or total moles, which includes the component. In a nonlimiting example, 10 grams of component in 100 grams of the material is 10 wt.% of that component in the material.[0049} As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, such as a monkey) and a non-primate (such as a cow, a dog, a horse, a sheep, a rabbit, a cat, a rat, or a mouse). In some aspects, the subject is a human. The subject can be an adult subject or a pediatric subject, such as a neonate, an infant, or a child. The subject can be a patient. A “patient” as used herein refers to a human individual who has or suspected to have a disease or condition, and / or who is a candidate for or benefits from a treatment for the disease or condition.

[0050] A nanoparticle is provided that delivers any therapeutic payload to any tissue with heretofore unknown efficiency and specificity. The nanoparticle can be used to prevent, treat, and cure all common and rare diseases. Associated methods and compositions are also provided.[0051| Mammary cells, for example and without limitation bovine mammary alveolar MAC- T cells or human mammary epithelial HME1 cells, are genetically engineered to secrete sEVs amenable to modification by chemistry protocols. A tetraspanin, such as CD81, CD9, and CD63, is firmly anchored in the sEV membrane and displays stretches of amino acids on the sEV surface. MAC-T cells or HME1 cells are engineered to substitute three distinct unnatural amino acids that do not naturally exist in mammalian proteomes, referred to herein as noncanonical amino acids (ncAAs), for natural amino acids on the sEV surface in tetraspanin (such as CD81, CD9, and CD63). In addition, CD81 is modified to express a poly-histidine tag, which enables the isolation of ncAA-containing sEVs from culture media by Ni2+affinity chromatography at great purity. The ncAAs are non-naturally occurring in the mammalian proteome and serve as unique targets for attaching synthetic peptides by bioorthogonal chemistry’.[0052| The binding of synthetic peptides to sEVs facilitates delivery of sEVs to target cells, tissues, or organs. One ncAA can be modified by attaching a peptide that decreases the elimination of sEV and therapeutic cargo by subject macrophages, such as CD24 or CD47. A second ncAA can be modified by attaching a peptide that steers sEVs to target tissues, such as apoE or CooP if brain tumors are the target. A third ncAA can be modified by attaching a peptide that decreases the rate of sEV degradation in target cell lysosomes, such as poly-arginine or Alix. The net result is that the concentration of the therapeutic sEV pay load is much higher in target tissues and much lower in non-target tissues compared to conventional nanomedicine protocols. sEVs may be loaded with any cargo, including CRISPR-Cas9 (gene editing), proteins and plasmids (gene delivery), and small interfering RNA (gene silencing), for targeted delivery.[0053 j Mammary cells such as MAC-T cells and HME1 cells secrete sEVs with a size, morphology, and bioavailability identical to sEVs from milk of the respective mammalian species (milk sEVs, MEVs). In MEVs secreted by genetically engineered mammary cells, such as MAC-T cells or HME1 cells provided herein, the three ncAAs in a tetraspanin (such as CD81, CD9, and CD63) can be modified with any peptide of choice in any combination of choice after isolating the histidine positive MEVs using Ni2+columns. By choice of homing peptide, the MEVs can be directed to any tissue of interest. The list of homing peptides is constantly growing, and, to date, 100 peptides have been identified that serve as homing signals for 20 tissues, all of which can be attached to MEVs provided herein via a ncAA as a homing peptide. If better peptides are discovered in future research, they can be attached to existing ncAAs in the MEV nanoparticle without the need to develop a new cell line. The MEVs provided herein evade elimination by macrophages and degradation in target cells. If, over time, a peptide elicits an antigenic response in subjects, the sEVs can be modified to evade degradation by the immune system by substituting peptides attached to ncAAs, such as apoE for CooP, or CD24 for CD47.[0054J The compositions and methods provided herein afford clinicians novel tools for treating diseases, including the rare diseases.A. Modified small extracellular vesicles[00551 A modified small extracellular vesicle (sEV) is provided. The modified sEV contain at least one tetraspanin, which contain at least one noncanonical amino acid (ncAA) substituting for a native amino acid in an extra-exosomal loop. The sEV can be derived from milk, for example without limitation, bovine milk, ovine milk, or caprine milk. The EVs can be derived from cells (such as a cell line), and can be of a size, morphology, and bioavailability identical to an sEV derived from milk (such as bovine milk, ovine milk, or caprine milk). These sEVs can be referred to as small extracellular vesicles (sEVs) from milk (MEVs). MEVs have qualities desired in nanomedicine such as oral bioavailability, ability to cross barriers such as placenta and the blood-brain barrier, protection of payload in the gastrointestinal tract and industrial processing, and undetectable toxicity and immunogenicity.sEVs express three tetraspanins (such as CD81 CD9, and CD63) in all possible permutations. The engineered MEVs provided herein have at least one tetraspanin that is modified such that, after purification, the MEVs have one or more (such as three) unique ncAAs as docking sites for distinct peptides. This is a major improvement compared to conventional protocols that insert lipid anchors in the sEV membrane, which may transfer to other lipophilic nanoparticles and do not allow to precisely control the stoichiometry of peptide binding. The tetraspanin containing ncAA can be selected from the group consisting of CD81, CD9, and CD63. In specific embodiments, the tetraspanin containing ncAA is CD81. One tetraspanin can containe 1, 2, 3, 4, or more ncAAs. In some embodiments, one tetraspanin contains three ncAAs substituting for three native amino acids.|(IO56] The ncAAs can include those with various functional groups, such as: an azide group, exemplified by p-azido-L-phenylalanine; a carbonyl group, as in 2-amino-8-oxononanoic acid; and strained alkenes, represented by trans-cyclooct-2-en-L-lysine. In specific embodiments, the noncanonical amino acid is p-azido-L-phenylalanine, 2-amino-8-oxononanoic acid, and N6-(3-(cyclobut-2-en-l-yl)propanoyl)lysine. For example, the noncanonical amino acid can be p-azido-L-phenylalanine, 2-amino-8-oxononanoic acid, and N6-(3-(cyclobut-2-en-l- yl)propanoyl)lysine.|0057] In specific embodiments, CD81 is modified to have three ncAAs. The absence of glycoproteins in CD81 ensures that ncAAs are not buried in MEV glycans when attaching synthetic peptides. The MEVs provided herein can be used for gene editing, gene delivery, and gene silencing.[0058| An embodiment of a genetically engineered MEV is depicted in FIG. 1A. The left panel of FIG. 1A shows an MEV having three distinct tetraspanins anchored in sEV membranes, namely CD9, CD63, and CD81. Mammary cells such as MAC-T cells and HME1 cells can be genetically engineered to substitute three distinct unnatural amino acids (noncanonical amino acids, ncAAs) for natural amino acids on the MEV surface in a tetraspanin (such as CD81), as shown in the right panel of FIG. 1A. Mammary cells (such as MAC-T cells and HME1 cells) may be immortalized, for example by stably expressing hTERT, as shown in FIG. 1A (“hTERT-HMEl”). A tetraspanin (such as CD81) can further be modified to express a cleavable poly-histidine tag (“His6” in FIG. 1A), which enables purification of MEVs from culture media by Ni2+affinity chromatography. Natural MEVs do not express a poly -histidine tag and are therefore be removed at the Ni2+affinity chromatography step. Following the purification of MEVs from culture media, the ncAAs serve as unique targets for attaching synthetic peptides by bioorthogonal (“click”) chemistry, followed by loading with therapeuticcargo. FIG. 3B depicts use of bioorthogonal (click) chemistry to insert a ncAA to a tetraspanin (such as CD63) for expression in cells. FIG. 3C depicts expression of a CD63-iRFP fusion protein containing azido-Phe in iRFP. As shown in FIG. 1A, the MEVs may be loaded with cargo, such as CRISPR-Cas9, guide RNA, proteins (such as therapeutic proteins), plasmids (such as plasmids encoding therapeutic proteins), and small interfering RNA.[0059} The ncAAs are non-naturally occurring in the mammalian proteome and, therefore, synthetic peptides do not bind anywhere else but to their complementary ncAAs in the tetraspanin (such as CD81). Table 1 show-s examples of ncAA, their reactive groups, and complementary' groups in synthetic peptides.Table 1. Reactive groups in ncAAs serve as binding sites for complementary groups in synthetic peptides[0060} In the MEVs provided herein, one ncAA can be modified by attaching a peptide, such as CD24 or CD47, that decreases the elimination of MEVs and therapeutic payload by macrophages of the subject to whom the MEVs are administered. A second ncAA can be modified by attaching a peptide that steers MEVs to the desired target tissues, such as peptides apoE or CooP if brain tumors are the target. A third ncAA can be modified by attaching a peptide that decreases the rate of MEV degradation in target cell lysosomes, such as polyarginine or Alix. The net result is that the concentration of the therapeutic MEV payload is much higher in target tissues and much lower in non-target tissues compared to conventional nanomedicine protocols. The sEV can further contain a poly-histidine tag in the tetraspanin. MEVs can be loaded with any cargo, including CRISPR-Cas9 and guide RNA, proteins (such as therapeutic proteins), plasmids (such as plasmids encoding therapeutic proteins), and small interfering RNA. The sEV can further contain a detectable label. The detectable label can contain a fluorophore and / or a pH-sensitive dye.10061} In specific embodiments, CD81 on MEVs are modified because CD81, unlike other tetraspanins such as CD9 and CD63, is not glycosylated. Absence of glycoproteins ensures that ncAAs are not buried in MEV glycans when attaching synthetic peptides. At least one aminoacid in at least one tetraspanin is modified. In specific embodiments, three amino acids in one tetraspanin are modified (as opposed to one amino acid in each of the three tetraspanins) because this approach ensures that all MEVs purified by Ni2+columns will contain three unique ncAAs. Without wishing to be bound by theory, sEVs constitute heterogenous populations of natural nanoparticles and not every sEV expresses all three tetraspanins. In some embodiments, mammary cells such as MAC-T cells or HME1 cells are engineered to produce modified MEVs. Additionally, animals, such as cows can be engineered to produce modified MEVs. [0062 J MEVs provided herein have characteristics suited for delivering therapeutic payloads to recipient cells. MEVs protect their cargo against degradation in the gastrointestinal tract and industrial processing. Animal studies show that about 50% of orally administered MEVs are absorbed, and oral bioavailability of MEV cargo (RNA) has been demonstrated in humans. In addition to crossing the intestinal barrier, MEVs also cross barriers like vascular endothelial cells, placenta, and the blood-brain barrier. The intestinal mucosa, liver, and brain are the primary accumulation sites of orally administered MEVs. Furthermore, MEVs are biologically safe. Studies have shown that MEVs are not immunogenic and do not elicit adverse reactions. They did not elicit a cytokine storm in human plasma following oral administration and in human peripheral blood mononuclear cells ex vivo. MEVs did not elicit an immune response or impair liver and kidney function and ery thropoiesis in rats. MEVs did not cause liver or kidney damage and did not alter food and water intake or physical activity in mice. Protocols are available for loading sEVs, including MEVs, with CRISPR-Cas9 and guide RNA (to edit genes), proteins and plasmids (to deliver genes), and small interfering RNA (to silence genes). Moreover, sEV payloads retain biological activity'. For example, ere recombinase included in sEVs caused recombination events in reporter mice. Chemotherapeutics, loaded into MEVs, slowed tumor growth and increased survival in murine xenograft models of human cancer. Mammary cells such as bovine mammary alveolar MAC-T cells and human mammary epithelial HME1 cells secrete MEVs and are amenable to genetic engineering. Mammary' cells such as MAC-T cells or HME1 cells are an excellent tool for optimizing experimental approaches in an economical cell culture-based system prior to developing transgenic animals (such as cows) that produce MEVs. Eighth, MEVs can be produced in large scale. A cow produces 10,800 kg milk annually and one milliliter of milk contains 1014sEVs, which equates to 1021MEVs per cow annually.|0063] FIG. IB schematically depicts sEV uptake, degradation, and retrofusion. Macrophages rapidly eliminate synthetic nanoparticles and sEVs. The capacity of MEV internalization is much greater in macrophages than any other cell type studies to date. Provided herein areMEVs that attach a Don’t-eat-me signal like CD24 or CD47. As provided herein, attachment of CD24 or CD47 to MEVs can cause a 50% decrease in MEV uptake by macrophages and an up to 12.3-fold decrease of MEV elimination in mice (see FIG. 8B, Example 3), leading to a decrease of MEV accumulation in resident macrophages such as Kupffer cells in the liver. Dual MEV modification by CD24 and C47 can have an additive or synergistic effect. The attachment of multiple peptides can have an additive or synergistic effect.100641 Nanoparticles do not accumulate in target tissues with great specificity unless modified by attaching a homing signal. Provided herein is a method to attach tissue-homing peptides to MEVs, and MEVs with tissue-homing peptides attached. MEVs provided herein can have any suitable homing peptides, including any of the about 100 peptides that have been identified to serve as homing signals for 20 tissues.[0065} More than 80% of sEVs are degraded in lysosomes upon internalization by target cells, thereby leading to a loss of therapeutic payloads. The alternative pathway, “retrofusion,” as used herein refers to the fusion of sEVs with the limiting membrane of the multi vesicular body, thereby releasing the payload into the cytosol. FIG. IB schematically depicts retrofusion. In some embodiments, MEVs provided herein have retrofusion signals such as poly-arginine (R16) attached. Decoration of sEVs with R16 increased by 75% the biological activity of the anti-cancer drug saporin in Chinese hamster ovary CEIO-K1 cells.

[0066] Nanoparticles and MEVs are covered by various proteins (“corona”) after entering biological environments, which may alter tissue homing. In some embodiments, MEVs provided herein have modifications that decrease the formation of corona such as cysteine, polyethylene glycol, and polyethylene oxide.[0067| Conventional strategies for using sEVs rely on expressing one transgene in cells, which then secrete modified sEVs into culture media, such as CD24. CD47, or R16-positive sEVs. This is a major limitation because a new cell line must be developed every time a peptide modification does not perform as desired, or a new improved peptide becomes available, or a peptide elicits an immune response in patients. In contrast, the present disclosure provides MEVs having three ncAAs that can be modified with any peptide of choice. For example, CD47 may be substituted for CD24 if CD24 causes an immune response. apoE may be substituted for CooP if brain tumors are the target and CooP does not meet expectations. Purification of MEVs from genetically engineered mammary cells such as MAC-T cells or HME1 cells by using Ni2+columns will ensure that three ncAAs are present in every MEV, as shown in FIG. 1A.

[0068] Another conventional approach is to insert lipid anchors in the sEV membrane, whichserve as binding sites for peptides. This approach has major limitations. First, lipophilic anchors and dyes transfer from the sEV membrane to other lipophilic nanoparticles. Second, it is difficult if not impossible to control the number of lipid anchors in the sEV membrane and the stoichiometry of distinct modifiers attached to the membrane. In contrast, the MEVs provided herein have tetraspanins with ncAAs for peptide binding sites. First, tetraspanins such as CD81. CD9, and CD63 are firmly anchored in the MEV membrane, such that the “anchor” does not transfer to other nanoparticles. Second, the linkage formed by bioorthogonal chemistry is stable under physiological conditions, such that the release of peptides is not a concern. Third, all MEVs purified from mammary cell culture media such as MAC-T or HME1 cell culture media will harbor three distinct ncAAs at a 1 : 1 : 1 ratio, thereby affording full control over the stoichiometry of peptide modifications.

[0069] sEV purity is an important consideration in the clinical translation of therapeutics- loaded sEVs because of the heterogeneity of sEVs composition, size, and batch-to-batch variation during production. For example, the presence of tetraspanins varies among individual sEVs and not every sEV expresses all three tetraspanins. Likewise, when working with genetically engineered cells or livestock, MEV preparations will be a mixture of genetically modified MEVs (CD81 transgene) and natural MEVs (endogenous CD81 gene). In the MEVs provided herein, the poly-histidine tag thereof enables separation of modified MEVs from natural MEVs, thereby enhanced purity.

[0070] The MEVs and related methods provided herein provide clinicians with a versatile tool for editing genes by CRISPR-Cas9 (prevention and cure of disease), delivering genes in form of plasmids and proteins (treatment of disease), and silencing genes by small interfering RNA (treatment of disease) in any tissue of choice (Figure 3). The MEVs provided herein enables preventing, treating, and curing disease in any tissue with any payload with heretofore unknown specificity. This applies to both common and rare diseases.

[0071] Also provided is a pharmaceutical composition containing a therapeutically effective amount of the sEV provided herein.

[0072] The carbohydrate trehalose is the gold standard of cryoprotection of sEVs in pharmaceutical applications. Trehalose has received Generally Recognized As Safe status from the FDA. As shown in FIG. 13, Purified MEs can be preserved during lyophilization through the addition of 5% trehalose, which improves MEV storage and facilitate the distribution of MEVs in regions w ith poor medical infrastructure.B. Engineered Cell Line, Engineered Animal, and Compositions for Producing Modified sEVs[0073| Mammary cells such as bovine mammary alveolar MAC-T cells and human mammary alveolar HME1 cells, immortalized by using SV-40 large T-antigen, secrete sEVs with a size, morphology, and bioavailability identical to sEVs from milk. Provided herein are monoclonal engineered mammary cells, such as mammary alveolar MAC-T cells or mammary epithelial HME1 cells in which an exosome protein contains three distinct ncAAs and a poly -histidine tag (Table 2). The exosome protein can be tetraspanin. In specific embodiments, the exosome protein that contains the three ncAAs is CD81. The ncAAs are non-naturally occurring in the mammary' cell (such as MAC-T cell or HME1 cell) proteome and will be inserted in the extra- exosomal loops in CD81 so that homing peptides are exposed on the outer MEV surface.Table 2. ncAAs in CD81 in MAC-T cell or HME1 cell-derived MEVs

[0074] The genome of the cell line can contain at least one amber mutation site encoding the at least one ncAA and a poly-histidine tag, to be expressed in a loop on the surface of the at least one tetraspanin, and at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin. In certain embodiments, the cell line can include a biosynthetic enzyme that converts at least one ncAA precursor to the at least one ncAA. For example, the genome of the cell line can contain three amber or quadruplet codon mutation sites, or a combination thereof, each encoding an ncAA. and a poly-histidine tag, to be expressed in a loop on the surface of CD81 , and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent CD81. In certain embodiments, the cell line can include three biosynthetic enzyme that convert the ncAA precursors to the three ncAAs. For example, the genome of the cell line can contain three amber and quadruplet codon mutation sites.|0075[ In some embodiments, ten genetic modifications are introduced in the mammary cell (such as MAC-T cell or HME1 cell) genome: three amber or quadruplet codon mutation sites, or combinations thereof, encoding ncAAs, and a poly -histidine tag in the small or large loopson the tetraspanin (such as CD81) surface (FIG. 2); and three aininoacyl-tRNA synthetase (aaRS) / tRNA pairs for incorporating ncAAs in nascent tetraspanin (such as CD81) (FIG. 3A).

[0076] Studies have established the use of genetic code expansion for inserting ncAAs in proteins in live cells (FIG. 3A). In this approach, aaRS / tRNA pairs, which do not cross-react with any of the endogenous tRNAs and aaRSs of the host, are engineered to recognize a codon that does not encode natural proteinogenic amino acids, such as the amber UAG codon. Using the UAG codon for inserting an ncAA is not a problem because the aaRS is modified to charge its cognate tRNA with only the desired ncAA. This methodology enabled the incorporation of over 300 ncAAs with a variety of side chain structures and functions in live cells, including spectroscopic probes, metal chelators, posttranslational modification analogs, redox-active groups, and probes with unique chemical reactivities. This methodology has been successfully applied in vertebrate animals including mice and zebra fish.

[0077] In some embodiments, the endogenous gene expression in mammary cells (such as MAC-T cells and HME1 cells) is left intact. In some embodiments, such as when a natural tetraspanin, such as natural CD81 is expressed more efficiently than the genetically altered CD81, and the presence of the endogenous CD81 could lead to a low expression of the altered CD81, the endogenous CD81 gene can be knocked out for example by using CRISPR-Cas9.|0078] Also provided is a plasmid containing a nucleic acid sequence encoding: at least one tetraspanin containing at least one amber mutation site encoding at least one noncanonical amino acid (ncAA), and a poly -histidine tag, to be expressed in a loop on the surface of the at least one tetraspanin, a biosynthetic enzyme that converts at least one ncAA precursor to the at least one ncAA, and / or at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin. In specific embodiments, the plasmid contains a nucleic acid sequence encoding: CD81 containing three amber or quadruplet codon mutation sites, or combinations thereof, each encoding an ncAA, and a poly-histidine tag, to be expressed in a loop on the surface of the at least one tetraspanin, and / or three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent tetraspanin. An example set of ncAAs, amber and quadruplet codons, and anti-codons (tRNA) is shown in Table 2.

[0079] Also provided is a transgenic animal capable of producing the sEV provided herein. The transgenic animal can be a cow, and the milk of the cow contains the sEV provided herein. A genome of the transgenic animal can contain at least one amber mutation site encoding the at least one ncAA, and a poly-histidine tag to be expressed in a loop on the surface of the at least one tetraspanin. a biosynthetic enzyme that converts at least one ncAA precursor to the at least one ncAA, and / or at least one aminoacyl-tRNA synthetase / tRNA pair for incorporatingthe at least one ncAA in a nascent tetraspanin. In some embodiments, the genome of the transgenic animal contains three amber or quadruplet codon mutation sites, or combinations thereof, each encoding an ncAA, and a poly-histidine tag to be expressed in a loop on the surface of CD81, and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent CD81. In some embodiments, the genome of the transgenic animal contains three amber and quadruplet codon mutation sites.C. Treating diseases using MEVs[0080 j Provided herein is a method of treating a subject having a disease. The method includes administering a therapeutically effective amount of the sEV or the pharmaceutical composition provided herein. In some embodiments, a peptide that steers the sEV to a diseased tissue is attached to the at least one ncAA. MEVs provided herein can be loaded with cargo, such as a therapeutic agent, and delivered to any tissue of interest, for example to treat disease or condition, including common diseases and rare diseases.

[0081] For example, the MEVs can be used to treat glioblastoma multiforme (GBM). Treatment of GBM using the MEVs serves as one example of how the MEVs provided herein can be applied in the treatment of a common disease with poor prognosis. Despite the availability of 13 Federal Drug Administration (FDA)-approved drugs to treat brain tumors, an estimated 18.000 of the 24,000 Americans diagnosed (75%) with malignant brain tumors died from the disease in 2020. GBM is the most common malignant intracranial tumor, accounting for approximately 30% of primary brain tumors, and has a median survival of only 10-14 months with no more than 5% of patients surviving more than three years. Peptides have been identified that direct nanoparticles to GBM tumors, such as the apoE or CooP peptides. MEVs provided herein, modified through the covalent attachment of apoE or CooP, accumulate in the brain and deliver their payloads to tumors. Payloads can target, for example, mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Mutations in IDH1 occur in the vast majority of glioma and the R132H mutation is the most common among IDH1 mutations. Examples of MEV pay loads include IDH1 siRNA to knock down the expression of the oncogenic mutant IDH1 allele (without affecting the expression of the wild-type allele) and CRISPR-Cas9 proteins and guide RNA to edit the mutant allele.

[0082] The MEVs provided herein can be used to treat heart disease. Heart disease is the leading cause of death for people in the U.S. and is poised to benefit from advances in nanomedicine. Approximately 655,000 Americans die from heart disease every year and costs related to the disease are high - roughly $219 billion annually. The use of RNA therapeuticsholds great promise in preventing and treating heart disease. In the context of cardiovascular disease, the use of RNA therapeutics is being explored in the treatment of hypercholesterolemia, hypertriglyceridemia, cardiac amyloidosis, and atrial fibrillation. Twenty RNA therapeutics for reducing risk factors leading to cardiovascular disease were undergoing testing in randomized clinical trials, Phases 1 through 3 in 2018, and five RNA treatments received FDA approval by 2018. A search of NIH’s ClinicalTrials.gov database using the search terms ‘‘heart disease” and “RNA” retrieved 837 clinical trials. The use of RNA therapeutics in the treatment of heart disease faces the same challenges as the use of RNA therapeutics to treat any other disease, including RNA degradation by circulating nucleases, non-target effects, and release of pro-inflammatory cytokines induced by siRNAs through tolllike receptor signaling pathways. Encapsulation of RNA therapeutics in MEVs modified with homing peptides eliminates or minimizes these challenges.

[0083] In addition to treating patients with common diseases such as GBM, Alzheimer’s disease, and pancreatic cancer, the MEVs provided herein can be used to treat an underserved group of patients, i.e., individuals suffering from rare diseases. Diseases are considered rare in the U.S. if affecting fewer than 200,000 patients. Collectively, rare diseases affect approximately 30 million people in the U.S. Well-known examples include cystic fibrosis (lung), Huntington’s disease (brain), Syngapl haploinsufficiency (brain), and Duchenne muscular dystrophy (muscle). As the majority of rare diseases arise fonn genetic disorders, gene therapy is a potentially rewarding option for treating and curing them. Treatment can be achieved by delivering mRNA, plasmids, or proteins to target tissues, whereas cure can be achieved by delivering gene-editing tools (CRISPR-Cas9 proteins and nucleic acids) to target tissues. For at least 24 rare diseases, the delivery' of RNA therapeutics, such as gene silencing by siRNA and gene editing by CRISPR-Cas9, to sites of disease is considered a viable approach to treatment and cure, including cystic fibrosis. Huntington’s disease, and Duchenne muscular dystrophy. Homing peptides are known for the sites in which these diseases reside, i.e., lung, brain, and muscle. The peptide-modified MEVs provided herein, loaded with RNA therapeutics, can treat these diseases and improve the quality of life of persons suffering from rare diseases without requiring major follow-up investments in developing disease-specific drugs.

[0084] For example, the MEVs provided herein can be used to treat Syngapl haploinsufficiency. Syngapl haploinsufficiency is a rare disease with an incidence of 1-4 in 10,000 individuals. Syngapl haploinsufficiency causes neurodevelopmental disorders defined by autistic traits, cognitive impairment and epilepsy. The MEVs provided herein can deliverthe Syngapl gene or a plasmid encoding Synga l to the brain (see Example 4, FIGs. 11A and 11B), thereby benefiting patients suffering from Syngapl haploinsufficiency.

[0085] The MEVs provided herein can be used to treat Magel2 associated disease. The MEVs provided herein can deliver the Magel2 gene or a plasmid encoding Magel2 to the brain (for example hypothalamus), thereby benefiting patients suffering from Syngapl haploinsufficiency. Magel2 associated disease can include, without limitation, Prader-Willi syndrome, Angelman syndrome, Schaaf-Yang Syndrome, and Distal Arthrogryposis With Hypopituitarism, Intellectual Disability, And Facial Anomalies.

[0086] Provided herein are combinations of a genetically engineered mammary cell line with bioorthogonal chemistry to produce MEVs for the prevention, treatment, and cure of diseases. First, conventional protocols attached a single homing peptide to nanoparticles, i.e., clinicians were not provided with the flexibility of the technology discussed here. The MEVs and methods provided herein allow the combination of homing peptide with Don ’t eat me peptides. This adds another layer of versatility because elimination of nanoparticles and sEVs by macrophages is a major challenge in nanomedicine. Second, conventional protocols insert lipid anchors in sEV membranes, which serve as binding sites for modifiers of choice. This approach has major limitations including the transfer of lipophilic anchors from the sEV membrane to other lipophilic nanoparticles and difficulties with controlling the stoichiometry' of distinct modifiers attached to lipid anchors. The MEVs and methods provided herein overcome these limitations because (1) tetraspanins such as CD81 are finnly anchored in the sEV membrane and will not transfer to other compounds; (2) the 1,2,3-triazole linkage formed by bioorthogonal chemistry7is stable under physiological conditions and therefore the release of peptide modifications is not a concern; and (3) all MEVs purified from mammary cells such as MAC-T or HME1 cell culture media will harbor three distinct ncAAs (peptide binding sites) at a 1: 1 : 1 ratio, thereby affording full control over the stoichiometry of peptide modifications. Third, conventional protocols use sEVs that are rapidly7degraded in lysosomes upon internalization by target cells. It has been estimated that more than 80% of sEVs are degraded in lysosomes within six hours of internalization, thereby leading to a loss of therapeutic payloads delivered by nanoparticles. The MEVs and methods provided herein allow the combination of homing peptides and Don ’t eat me peptides with peptides that enhance MEV retrofusion, thereby enhancing MEV survival and increasing the concentration of therapeutic payloads in target cells. Finally, a poly-histidine tag in MEVs enables the isolation of “pure” ncAA-containing MEVs from mammary cells such as monoclonal MAC-T or HME1 cell lines.

[0087] Due to the versatile nature of the genetically altered MEVs, the MEVs provided hereincan be used in the prevention, treatment, and cure of disease in which the delivery of therapeutic payloads by nanoparticles is desirable and tissue-homing peptides are available. The MEVs are useful for common (such as GBM, cardiovascular disease, Alzheimer’s disease) and rare diseases.D. Producing modified small extracellular vesicles

[0088] Provided herein is a method of producing a modified small extracellular vesicle (sEV). The method includes introducing into a genome of a cell line at least one amber mutation site encoding the at least one ncAA, and a poly -histidine tag to be expressed in a loop on the surface of the at least one tetraspanin, a biosynthetic enzyme that converts at least one ncAA precursor to the at least one ncAA. and / or at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin; culturing the modified cell line in a cell culture medium; and isolating the sEV from the cell culture medium. In some embodiments, the cell line is a mammary cell, such as a mammary alveolar MAC-T cell line or a mammary epithelial HME1 cell line.

[0089] Also provided herein is a method of producing a modified small extracellular vesicle (sEV). The method includes introducing into a genome of an animal at least one amber mutation site encoding the at least one ncAA, and a poly -histidine tag to be expressed in a loop on the surface of the at least one tetraspanin, a biosynthetic enzyme that converts at least one ncAA precursor to the at least one ncAA, and / or at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin; collecting milk produced by the animal; and isolating the sEV from the milk. The animal can be a ruminant, such as a cow.

[0090] The methods can include introducing into the genome three amber or quadruplet codon mutation sites, or combinations thereof, each encoding an ncAA, and a poly -histidine tag, to be expressed in a loop on the surface of CD81, and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent CD81.

[0091] The ncAAs can include those with various functional groups, such as: an azide group, exemplified by p-azido-L-phenylalanine; a carbonyl group, as in 2-amino-8-oxononanoic acid; and strained alkenes, represented by trans-cyclooct-2-en-L-lysine. In some embodiments, the three ncAAs include one or more of p-azido-L-phenylalanine, 2-amino-8-oxononanoic acid, and N6-(3-(cyclobut-2-en-l-yl)propanoyl)lysine.

[0092] The method can further include attaching a peptide that decreases the elimination of sEV by macrophages to a first ncAA, attaching a peptide that steers the sEV to a target tissueto a second ncAA, and / or attaching a peptide that decreases the rate of degradation of the sEV by target cell lysosomes to a third ncAA. The peptide that decreases the elimination of sEV by macrophages can include CD24 or CD47, the peptide that steers the EV to the target tissue can include apoE or CooP, and / or the peptide that decreases the rate of degradation of the sEV by target cell lysosomes can include poly -arginine or Alix.EXAMPLESExample 1: Genetic engineering of bovine mammary alveolar MAC-T cells

[0093] MAC-T cells were genetically engineered to express a ncAA. / ?-azido-L-phenylalanine. in CD63 fused with near-infrared protein (iRFP). Cell cultures were supplemented with a precursor of / ?-azido-L-phenylalanine. 4-azidobenzoic acid. If there is no incorporation of p- azido-L-phenylalanine, the amber codon (“TAG” in FIGs. 5A, 5B) functions as a stop codon, and iRFP is not expressed (therefore no fluorescence). iRFP fluorescence was observed herein, suggesting that >-azido-L-phenylalanine was inserted during the translation of the CD63-iRFP fusion protein (FIG. 5C). Two distinct amber codon sites, one replacing the tryptophan in position 126 in CD63, and the other replacing the proline in position 158, were evaluated (FIG. 5C). When the concentrations of / ?-azido-L-phenylalanine (AzF) were increased to 3 mmol / L or higher, toxicity was observed. However, 1 mmol / L or lower concentrations of ncAA can be used. Negative control cells were not transformed with the CD63-iRFP fusion construct and did not emit fluorescence (FIG. 5C). MAC-T cells not transfected with p-azido-L- phenylalanine tRNA were used as negative control and did not emit fluorescence (FIG. 5C). Cells transfected with a plasmid coding for CD63-iRFP were used as positive control, and emitted fluorescence (FIG. 5C).[0094} tRNA and aaRS pairs that recognize desirable ncAAs are inserted into the bovine ROSA26 locus in MAC-T cells. The expression of aaRS and tRNA is under the control of the human ubiquitin C promoter which was effective in MAC-T cells as disclosed herein. Amber mutations and a poly-histidine tag (His 10) is introduced into predefined sites in CD81 using a CRISPR / Cas9-based approach. RT-PCR is used to confirm the presence of aaRS and tRNA transcripts. MAC-T cells is genetically engineered to acquire the ability to synthesize ncAAs from non-toxic and low-cost carboxylic acids provided through culture media (Table 3). The engineered cells have the ability to convert the carboxylic acids to the desired ncAAs in a four- step metabolic pathway. The chemical modifications do not occur spontaneously or in any other site than MEVs because the ncAAs and complementary groups are unique to MEVs and the synthetic peptides (Table 1). Clones are isolated and expanded by using a CellRaft AIR®System (Cell Microsystems, Inc.). The site-specific incorporation of ncAAs in CD81 is confirmed by mass spectrometry. An amber mutation is inserted by substituting p-azido-L- phenylalanine (AzF) for phenylalanine-158 in CD63 fused with near-infrared fluorescent protein (iRFP) in AzFRS / tRNA-positive MAC-T cells. The amber mutation serves as a stop codon in the absence of AzF and translation of the fluorescent fusion protein stops prematurely (FIGs. 5A, 5B; FIG. 3C). If cells are provided with AzF, full-length CD63-iRFP (containing AzF in position 158) is translated and emits fluorescence (FIG. 3C). When culture media were supplemented with graded levels of AzF, cells emitted fluorescence (FIG. 5C).Table 3. Precursors and genes and tRNAs key to ncAA insertionExample 2: MEV purification, peptide attachment, and / or labeling[0095J For attaching peptide modifications, MEVs are purified from MAC-T cell cultures by using Ni2 tlarge scale resin using non-denaturing conditions (complete resin, Roche. In.), and authenticated by nanoparticle tracking analysis (NT A) (as shown in FIG. 4A), transmission electron microscopy (TEM) (as shown in FIG. 4B), and immunoblot analysis as recommended by the International Society for Extracellular Vesicles and shown above. If preparations are contaminated with sEVs and nanoparticles other than MEVs, samples are pre-purified by using size exclusion chromatography or iodixanol gradient centrifugation prior to Ni2+chromatography. Peptides with C-terminal alkyne, alkoxyamine, and tetrazine moieties are chemically synthesized in a commercial facility (such as CHI Scientific) and purity and identity are assessed by HPLC and mass spectrometry’. The peptides will be conjugated to ncAAs in MEVs as previously described.[0096| For studies of MEV elimination by macrophages and homing to target tissues, MEVs are labeled with fluorescent dyes by using a dye that emits fluorescence in the far-red range and is suitable for conjugation to MEVs by bioorthogonal reactions, such as MB 660R (Fisher Scientific, Inc.). For MEVs having only one ncAA, which is needed for attaching peptides, MEVs are labeled with a fluorophore that binds to carbonyl moieties on the MEV surface andemits fluorescence in the far-red range, such as HiLyte Fluor 750 (Anaspec, Inc.). Dyes that emit fluorescence in the far-red range have physicochemical properties superior to traditional dyes such as eGFP, such as deep tissue penetration, minimal photodamage, and minimal interference by background autofluorescence. The use of MB 660R and HiLyte Fluor 750 prevents the transfer of label from MEVs to other vesicles and complexes, which is a confounder in studies using lipophilic dyes. Also, HiLyte Fluor 750 is not released from MEVs in the gastrointestinal tract and plasma, i.e., we will not track detached dye (as opposed to labeled MEVs). Whether MB 660R is released from MEVs incubated in plasma, and artificial stomach and intestinal fluid, is assessed.[00971 Briefly, unmodified MEVs were loaded with a plasmid encoding iRFP, driven by CMV (FIG. 6A) by using electroporation and delivered to C57BL / 6J mice by nasal spray (3.366 / g body wt, once daily for seven days). Brains were harvested and fluorescence was assessed in a Li-COR small animal imaging system. iRFP was expressed, suggesting that the plasmid was released from MEVs into the cytosol and translated (FIG. 6B).[0098} For studies of MEV degradation in lysosomes, a pH-sensitive dye that covalently binds to amines on the MEV surface, pHrodo™ iFL Red STP Ester, is used. Fluorescence decreased to background levels as pHrodo-labeled MEVs were suspended in buffers of increasing pH (FIGs. 7A, 7B) When pHrodo-labeled MEVs were internalized by mouse bone marrow- derived macrophages (BMDMs), fluorescence was detected in acidic cytosolic organelles, most likely lysosomes (FIG. 7C).Example 3: MEV elimination by macrophages[0099} The accumulation of fluorophore-labeled sEVs, including MEVs, in plasma and tissues peaks at approximately 6 and 12 hours, respectively, following the administration of 1012sEVs per gram body weight by oral gavage in mice.

[0100] MAC-T cells were genetically engineered to secrete bovine MEs (hereafter MAC-T MEVs) decorated with a Don ’t-eat-me peptide. CD24 or CD47 by stably expressing the peptides in MAC-T cells. Ct values in transformed MAC-T cells were ~17 cycles for both CD24 and CD47, whereas Ct values were >35 (not detectable) in non-transformed controls (FIG. 8A). CD47-positive and CD47-negative MAC-T MEVs were purified from culture media conditioned with exosome-depleted fetal bovine serum) by ultracentrifugation. The MAC-T MEVs were labeled with a carbonyl-reactive dye that fluoresces in the near-infrared range, HiLyte 750™. The labeled MAC-T MEVs were administered to C57BL / 6 mice by nasal spray. The nasal route was chosen because the MAC-T MEVs have only to cross a thin layerof cells (olfactory bulb) adjacent to the nostrils for crossing the blood-brain barrier. Also, nasal sprays is anon-invasive approach for delivering therapeutics, encapsulated in MEs, to patients. Brains were dissected, perfused, and tissue accumulation of MEVs was assessed eight hours after MAC-T BME administration. CD47 had a strong, positive effect on the accumulation of MAC-T MEVs in the brain (FIG. 8C).

[0101] The increased accumulation of CD47-positive MEVs in the brain can be attributed to an increased concentration of CD47-positive MEVs in serum (slow elimination by macrophages). For example, the internalization of CD24 or CD47-positive MAC-T MEVs was approximately 50% lower compared CD24 / CD47-negative MAC-T MEVs in murine bone marrow derived macrophages (FIG. 8B). When CD24 and CD47 positive HiLyte-labeled MAC-T MEVs were administered by tail vein injection (1.5xl0n / g body wt.), and serum fluorescence was measured at t = 2 hours, serum values were 4.6 times and 12.3 times higher, respectively, as compared to unmodified MEVs: 65,619,236±39,871,170 for CD24, 175,710,383±45,580,988 for CD47, and 14,250,318±12,276,430 for controls (mean ± SEM, arbitrary units; 0.1<F<0.2: N=3).[0102| The effect of the attachment of Don 't eat me peptides, CD24 and / or CD47, in causing a decrease of MEV elimination by macrophages is evaluated. Briefly, MEVs are functionalized by attaching synthetic CD24 or CD47 peptides to ncAAs in purified MEVs, followed by labeling with HiLyte Fluor 750 or MB 660R. Possible position effects by attaching CD24 and CD47 to all three ncAAs in CD81 , and synergistic effects of CD24 and CD47 by attaching both peptides, are assessed. Studies are conducted in male and female C57BL / 6J mice ages five to eight weeks (Jackson stock no. 000664).Example 4: Tissue homing of MEVs[0103| Plasmids, delivered by MEVs, were expressed in the murine brain. Plasmids delivered by MEVs were expressed in the brain in Syngapl mutant mice, a model for a gene mutation that impairs cognition and speech. Briefly, MEVs (not modified by CD24, CD47, or RVG) were loaded with a CMV-CD63 / iRFP plasmid encoding enhanced green fluorescent protein (EGFP) or iRFP (FIG. 6A) with the using electroporation and delivered to C57BL / 6J mice by nasal spray (3.366 / g body wt, once every other day for 14 days). The mice expressed both EGFP and iRFP in the brain, whereas no fluorescence was detected in mice treated with plasmid-free MEVs. FIG. 6B shows the expression of iRFP without densitometry analysis (left) and with denistometry analysis (right).

[0104] MAC-T cells were genetically engineered to express CD63 fused with homing peptidesfor GBM (apoE and CooP) and healthy brain (RVG). When MAC-T MEVs were decorated with brain tumor homing peptides. apoE and CooP, the uptake of MAC-T MEVs in glioma cell cultures increased. CD63-apoE and CD63-CooP positive MAC-T MEVs were purified from conditioned media, labeled with HiLyte 750, and cultured with IDH1(R132H) glioma cells. The uptake of modified MAC-T MEVs was 40-50% greater than WT controls (FIG. 9), suggesting that peptide modifications can enhance the homing of MEV s to gliomas in patients and mice. Indeed, when CD63-apoE or CD63-CooP positive MEVs tagged with Al exaFluor 488 were administered intravenously to a mouse xenograft model of human GBM, the MEVs accumulated in tumors (FIG. 10).[0105 j Next, MAC-T cells were engineered to secrete MEVs decorated with the core peptide from the brain homing protein, RVG. MEVs were purified, labeled, and admistered as described above. When RVG-positive MEVs were loaded with a mammalian Syngapl expression plasmid using electroporation using electroporation and delivered as nasal spray, the expression of Syngapl mRNA and protein increased in the brain in SyngapT1' mice compared to RVG-free controls (FIGs. 11A and 11B). Importantly, the accumulation of RVG- positive MAC-T MEVs in tissues other than the brain was less than than that of RVG-negative MAC-T MEVs. i.e., the RVG modification reduces non-target effects.

[0106] Previous studies suggest that the accumulation of MEVs is low in the pancreas and heart (in contrast to major accumulation sites in the intestinal mucosa, liver, spleen, lungs, and brain). Accordingly, MEV homing to the heart and pancreas using the methods and compositions disclosed herein is evaluated.

[0107] Heart-homing (GGGVFWQ) and pancreas -homing (CHVLWSTRC) peptides are conjugated to ncAAs in CD81 in purified MEVs. MEVs are labeled with fluorophores as described above and administered by oral gavage to male and female mice using a doseresponse, time-course design as described above. MEV accumulation in perfused target tissues (heart or pancreas) and non-target tissues (intestinal mucosa, liver, spleen, lungs, and brain) are studied. As discussed for CD24 and CD47, position effects and additive effects for heart- and pancreas-homing peptides are assessed. The effect of the attachment of CD24 or CD47 to one of the three ncAAs, in addition to heart- or pancreas-homing peptides, in increasing MEV accumulation in the heart or pancreas is evaluated. Scrambled peptides and fluorophore-free MEVs will serve as controls.Example 5: Retrofusion

[0108] Putative retrofusion signals, poly-arginine and Alix are attached to MEVs, and theireffect is assessed as to increasing the release of MEV and their therapeutic payload from the multivesicular body into the cytosol, thereby increasing the concentration of therapeutic cargo in target cells.|0109] A mammalian iRFP reporter plasmid (FIG. 6A) and a pH-sensitive dye can be used for assessing MEV localization to lysosomes (FIG. 6B).

[0110] R16 and the N-terminal functional domain in Alix (responsible for interacting with TsglOl) are conjugated to ncAAs in CD81 in purified MEVs. Alternatively, the C-terminal domain in Alix can be conjugated to ncAAs in CD81 in purified MEVs. Two labels are used in separate experiments, conducted in parallel. In one line of experimentation, MEVs are labeled with pHrodo as described above, to identify the localization of MEVs to lysosomes. In a second line of experimentation, MEVs are loaded with the iRFP reporter plasmid, to identify the release of payloads into the target cell cytosol.

[0111] MEVs are added to cultures of murine BMDMs. Effects of Alix and R16 on the degradation of both MEVs and iRFP plasmid are assessed by using dose-response, time-course studies like those described for CD24 and CD47. The concentrations mimic MEV concentrations in human plasma (1.5xl0nMEVs / mL) and dilutions thereof (0.75xl0nand 0.15xl0nMEVs / mL). MEV degradation is assessed by using pHrodo accumulation in lysosomes, and iRFP plasmid retention is assessed by using near-infrared fluorescence as readout. Lysosomal localization of MAC -T MEVs and siRNA is identified by using confocal microscopy and staining lysosomes with anti-LAMP2. As control, cells treated with an inhibitor of lysosome acidification, chloroquine, are used. (1) position effects of peptides in CD81, (2) additive effects of the same modification, and (3) synergistic effects of both modifications are assessed. Scrambled peptides and plasmid-free MEVs serve as controls.[01121 The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

[0113] Other objects, features and advantages of the disclosure will become apparent from the foregoing figures, detailed description, and examples. It should be understood, however, that the figures, detailed description, and examples, while indicating specific embodiments of the disclosure, are given by way of illustration only and are not meant to be limiting. Additionally, it is contemplated that changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from the detailed description. In further embodiments, features from specific embodiments may be combined with features from other embodiments. For example, features from one embodiment may be combined with features from any of the other embodiments. In further embodiments, additional features may be addedto the specific embodiments described herein.Example 6: Storage stability of MEVs[0114| MEVs were purified from raw cow's milk from dairy herd by sequential ultracentrifugation. MEV count and size were assessed in freshly isolated MEs. and samples collected at timed intervals during storage at -80°C. The content of MEs decreased by 70±13% in samples stored at -80°C for two months compared to fresh samples and continued to decrease over time (FIG. 12).[0115| The carbohydrate trehalose is the gold standard of cryoprotection of sEVs in pharmaceutical applications. Trehalose has received Generally Recognized As Safe status from the FDA. As shown in FIG. 13, Purified MEs can be preserved during lyophilization through the addition of 5% trehalose, which improves MEV storage and facilitate the distribution of MEVs in regions with poor medical infrastructure.

Claims

CLAIMSWhat is claimed is:

1. A small extracellular vesicle (sEV) comprising at least one tetraspanin, the at least one tetraspanin containing at least one noncanonical amino acid (ncAA) substituting for a native amino acid in an extra-exosomal loop.

2. The sEV of claim 1, wherein the sEV is derived from mammalian milk, or comprise a size, morphology, and bioavailability identical to an sEV derived from mammalian milk.

3. The sEV of claim 1 or 2, wherein the at least one tetraspanin is selected from the group consisting of CD81. CD9. and CD63.

4. The sEV of claim 3, wherein the at least one tetraspanin is CD81.

5. The sEV of any one of claims 1-4, wherein the at least one ncAA comprises an azide group, a carboxyl group, or a strained alkene.

6. The sEV of claim 5, wherein the at least one noncanonical amino acid is selected from the group consisting of / i-azido-L-phenylalanine. 2-amino-8-oxononanoic acid, trans-cyclooct-2-en-L-lysine, and N6-(3-(cyclobut-2-en-l -yl)propanoyl)lysine.

7. The sEV of any one of claims 1-6, wherein the at least one tetraspanin comprises three ncAAs substituting for three native amino acids.

8. The sEV of claim 7, wherein the three ncAAs comprise -azido-L- phenylalanine, 2-amino-8-oxononanoic acid, and N6-(3-(cyclobut-2-en-l- yl)propanoyl)lysine.

9. The sEV of claim 7 or 8, wherein a peptide that decreases the elimination of sEV by macrophages is attached to a first ncAA, a peptide that steers the sEV to a target tissue is attached to a second ncAA. a peptide that decreases the rate of degradation of the sEV by target cell lysosomes is attached to a third ncAA, or a combination thereof.

10. The sEV of claim 9, wherein the peptide that decreases the elimination of sEV by macrophages comprises CD24 or CD47, the peptide that steers the EV to the target tissuecomprises apoE or CooP, the peptide that decreases the rate of degradation of the sEV by target cell lysosomes comprises poly-arginine or Alix, or a combination thereof.

11. The sEV of any one of claims 1-10, wherein the at least one tetraspanin further comprises a poly -histidine tag.

12. The sEV of any of claims 1-11, wherein the sEV is loaded with cargo.

13. The sEV of claim 12, wherein the cargo comprises a gene editing reagent, a protein, a plasmid, or a small interfering RNA.

14. The sEV of claim 13, wherein the gene editing reagent comprises a CRISPR- Cas9 enzy me and a guide RNA.

15. The sEV of claim 13, wherein the plasmid encodes Syngapl or Magel2.

16. The sEV of any one of claims 1-15, further comprising a detectable label.

17. The sEV of claim 16, wherein the detectable label comprises a fluorophore or a pH-sensitive dye, or a combination thereof.

18. A cell line capable of producing the sEV of any one of claims 1-17.

19. The cell line of claim 18, wherein the cell line is a mammary7alveolar MAC-T cell line or a human mammary epithelial EIME1 cell line.

20. The cell line of claim 18 or 19, wherein a genome of the cell line comprises at least one amber mutation site encoding the at least one ncAA, and a poly -histidine tag. to be expressed in a loop on the surface of the at least one tetraspanin, and at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin.

21. The cell line of claim 20, wherein the genome of the cell line comprises three amber and quadruplet codon mutation sites each encoding an ncAA, and a polyhistidine tag, to be expressed in a loop on the surface of CD81, and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent CD81.

22. A plasmid comprising a nucleic acid sequence encoding: at least one tetraspanin comprising at least one amber mutation site encoding at least one noncanonical amino acid (ncAA), and a poly-histidine tag, to be expressed in a loop on the surface of the at least one tetraspanin, and at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin.

23. The plasmid of claim 22, comprising a nucleic acid sequence encoding:CD81 comprising three amber and quadruplet codon mutation sites each encoding a noncanonical amino acid (ncAA), and a poly-histidine tag, to be expressed in a loop on the surface of the at least one tetraspanin, and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent tetraspanin.

24. A transgenic animal capable of producing the sEV of any one of claims 1-17.

25. The transgenic animal of claim 24, wherein the transgenic animal is a cow, wherein the milk of the cow comprises the sEV of any one of claims 1-17.

26. The transgenic animal of claim 23 or 24, wherein a genome of the transgenic animal comprises at least one amber mutation site encoding the at least one ncAA, and a poly -histidine tag to be expressed in a loop on the surface of the at least one tetraspanin, and at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin.

27. The transgenic animal of claim 26. wherein the genome of the transgenic animal comprises three amber and quadruplet codon mutation sites each encoding an ncAA, and a polyhistidine tag to be expressed in a loop on the surface of CD81, and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent CD81.

28. A pharmaceutical composition comprising a therapeutically effective amount of the sEV of any one of claims 1-17.

29. A method of treating a subject having a disease, the method comprising administering a therapeutically effective amount of the sEV of any one of claims 1-17 or the pharmaceutical composition of claim 28.

30. The method of claim 29, wherein a peptide that steers the sEV to a diseased tissue is attached to the at least one ncAA.

31. The method of claim 29 or 30, wherein the disease is glioblastoma multiforme, and the peptide that steers the sEV to the diseased tissue comprises apoE or CooP.

32. The method of claim 29 or 30, wherein the disease is Syngapl haploinsufficiency, and the sEV comprises a plasmid encoding Syngapl.

33. The method of claim 29 or 30, wherein the disease is a Magel2 -related disease, and sEV comprises a plasmid encoding Magel2.

34. The method of claim 29 or 30, wherein the disease is selected from the group consisting of Alzheimer's disease, pancreatic cancer, heart disease, cystic fibrosis, Huntington’s disease, and Duchenne muscular dystrophy.

35. A method of producing a modified small extracellular vesicle (sEV), the method comprising:(i) introducing into a genome of a cell line at least one amber mutation site encoding the at least one ncAA, and a polyhistidine tag to be expressed in a loop on the surface of the at least one tetraspanin, and at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin;(ii) culturing the modified cell line in a cell culture medium; and(iii) isolating the sEV from the cell culture medium.

36. The method of claim 35, wherein the cell line is a mammary alveolar MAC-T cell line or a human mammary epithelial HME1 cell line.

37. A method of producing a modified small extracellular vesicle (sEV), the method comprising:(i) introducing into a genome of an animalat least one amber mutation site encoding the at least one ncAA, and a polyhistidine tag to be expressed in a loop on the surface of the at least one tetraspanin, and at least one aminoacyl-tRNA synthetase / tRNA pair for incorporating the at least one ncAA in a nascent tetraspanin;(ii) collecting milk produced by the animal; and(iii) isolating the sEV from the milk.

38. The method of claim 37, wherein the animal is a cow.

39. The method of any one of claims 35-38, comprising introducing into the genome three amber and quadruplet codon mutation sites each encoding an ncAA, and a polyhistidine tag, to be expressed in a loop on the surface of CD81, and three aminoacyl-tRNA synthetase / tRNA pairs for incorporating the three ncAAs in a nascent CD81.

40. The method of claim 39, wherein at least one of the three ncAAs comprises an azide group, a carboxyl group, or a strained alkene.

41. The method of claim 40, wherein the three ncAAs comprise / i-azido-L- phenylalanine, 2-amino-8-oxononanoic acid, and N6-(3-(cyclobut-2-en-l-yl)propanoyl) lysine.

42. The method of any one of claims 39-41, further comprising: attaching a peptide that decreases the elimination of sEV by macrophages to a first ncAA, attaching a peptide that steers the sEV to a target tissue to a second ncAA, or attaching a peptide that decreases the rate of degradation of the sEV by target cell lysosomes to a third ncAA.

43. The method of claim 42, wherein the peptide that decreases the elimination of sEV by macrophages comprises CD24 or CD47, the peptide that steers the EV to the target tissue comprises apoE or CooP, or the peptide that decreases the rate of degradation of the sEV by target cell lysosomes comprises poly-arginine or Alix.

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