Engineered Extracellular Vesicles and Their Uses

Abstract

The present disclosure relates to therapeutic exosomes enriched with proteins present on the luminal surface of exosomes. The present disclosure provides methods for manufacturing exosomes enriched with proteins present on the luminal surface of exosomes, methods for associating therapeutic peptides or proteins with the luminal surface of exosomes, and methods of use, e.g., methods for therapeutic or diagnostic use. The manufacturing methods include generating lumen surface-engineered exosomes that comprise one or more of EV (e.g., exosome) proteins, modifications or fragments of EV (e.g., exosome) proteins, or fusion proteins of EV (e.g., exosome) proteins with payloads (e.g., bioactive molecules such as therapeutic proteins) at concentrations higher than those observed in wild-type exosomes.

Description

[0001] Reference to a Sequence Listing Submitted Electronically via EFS-WEB

[0002] The content of the electronically submitted Sequence Listing (name: 4000_041PC01_SL_ST25.txt, size: 116,344 bytes; and creation date: May 22, 2019) submitted in this application is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure provides extracellular vesicles (e.g., exosomes) enriched in scaffold proteins associated with the luminal surface of extracellular vesicles, which can be used as agents for preventing or treating cancer and other diseases. Background Art

[0004] Exosomes are important mediators of intercellular communication. They are also important biomarkers in the diagnosis and prognosis of many diseases, such as cancer. As drug delivery vehicles, exosomes offer many advantages over conventional drug delivery methods in many therapeutic areas as a new therapeutic modality.

[0005] A major characteristic of exosomes is their ability to contain bioactive payloads within their internal space or lumen. It is well known that exosomes contain endogenous payloads, including mRNA, miRNA, DNA, proteins, carbohydrates, and lipids, but the ability to direct the specific loading of a desired therapeutic payload is currently limited. Exosomes can be loaded by overexpressing the desired therapeutic payload in producer cells, but the efficiency of this loading is usually limited due to the random localization of the payload to the extracellular exosome processing centers. Alternatively, purified exosomes can be loaded ex vivo, for example, by electroporation. The efficiency of these methods can be very low or limited to small payloads, such as siRNA. Thus, there is a need for suitable methods for generating highly efficient and well-defined loaded exosomes to better realize the therapeutic uses and other applications of exosome-based technologies. Summary of the Invention

[0006] Aspects of the present disclosure relate to novel methods for loading extracellular vesicles (EVs) (e.g., exosomes) for therapeutic use. Specifically, the methods use newly identified protein markers from the luminal surface of exosomes. In particular, a group of proteins has been identified (e.g., myristoylated alanine-rich protein kinase C substrate (MARCKS); myristoylated alanine-rich protein kinase C substrate-like protein 1 (MARCKSL1); and brain acid-soluble protein 1 (BASP1)) that are highly enriched on the luminal surface of exosomes. In addition, it has been shown that a short sequence at the amino terminus of BASP1, e.g., at least seven amino acids, is sufficient to direct the efficient loading of fluorescent protein molecules to the same extent as the full-length BASP1 protein. This fragment is at least 7 amino acids and less than 10 amino acids, representing a significant advancement in the field of engineered EV loading and allowing for the efficient and reproducible loading of any bioactive molecule (e.g., therapeutic protein payload) into the lumen of EVs (e.g., exosomes) without additional ex vivo manipulation steps. Compared to any other genetic engineering method described to date, loading EVs (e.g., exosomes) using the fusion proteins described herein results in engineered EVs (e.g., engineered exosomes) with significantly higher payload levels.

[0007] The newly identified protein and peptide sequences from exosomes are used in various embodiments of the present disclosure. For example, some embodiments involve generating fusion proteins by conjugating EV (e.g., exosome) proteins or protein fragments (i.e., scaffold proteins) and bioactive molecules (e.g., therapeutically relevant proteins), and generating EVs (e.g., exosomes) that contain the fusion proteins on the luminal surface of the EVs. The bioactive fragments of native full-length proteins or bioactive molecules (e.g., therapeutically relevant proteins) can be transported to the luminal surface of EVs (e.g., exosomes) by conjugating them to exosome-enriched proteins or protein fragments.

[0008] The present disclosure also relates to the production or use of lumen-engineered EVs (e.g., lumen-engineered exosomes) that are designed for more efficient loading or for loading bioactive molecules (e.g., therapeutically relevant proteins) into the lumen of EVs (e.g., exosomes). For example, the luminal surface of EVs can be modified to contain a higher concentration of native full-length EV (e.g., exosome) proteins and / or fragments or modified proteins of native EV (e.g., exosome) proteins on the luminal surface.

[0009] Some embodiments of the present disclosure relate to producer cells or methods of generating such producer cells to produce such lumen-engineered EVs. An exogenous polynucleotide can be introduced transiently or stably into the producer cells to produce lumen-engineered EVs (e.g., lumen-engineered exosomes) from the producer cells.

[0010] Accordingly, on the one hand, the present disclosure provides EVs (e.g., exosomes) comprising a scaffolding protein, wherein at least a portion of the scaffolding protein is expressed from an exogenous sequence, and the scaffolding protein comprises MARCKS, MARCKSL1, BASP1, or a fragment, variant, derivative, or modification thereof.

[0011] In some aspects, the scaffolding protein is present in EVs (e.g., exosomes) at a higher density compared to different scaffolding proteins in different EVs (e.g., exosomes), wherein the different scaffolding proteins comprise conventional EV (e.g., exosome) proteins or variants thereof. In some embodiments, the conventional EV (e.g., exosome) proteins are selected from the group consisting of: CD9, CD63, CD81, PDGFR, GPI-anchored proteins, lactadherin, LAMP2, LAMP2B, and fragments thereof.

[0012] In some embodiments, the EVs (e.g., exosomes) are produced by cells genetically modified to contain an exogenous sequence, optionally wherein the cells are HEK293 cells.

[0013] In some embodiments, the cells contain a plasmid comprising the exogenous sequence.

[0014] In some embodiments, the exogenous sequence is inserted into a genomic locus that is 3' or 5' relative to the genomic sequence encoding MARCKS, MARCKSL1, or BASP1. In some embodiments, the exogenous sequence is inserted into the genomic sequence encoding MARCKS, MARCKSL1, or BASP1.

[0015] In some embodiments, the scaffolding protein is a fusion protein comprising MARCKS, MARCKSL1, BASP1, or a fragment thereof and a therapeutic peptide.

[0016] In some embodiments, the bioactive molecule comprises a therapeutic peptide selected from the group consisting of: a natural peptide, a recombinant peptide, a synthetic peptide, or a linker attached to a therapeutic compound. In some embodiments, the bioactive molecule (e.g., therapeutic compound) is selected from the group consisting of: nucleotides, amino acids, lipids, carbohydrates, and small molecules. In some embodiments, the bioactive molecule (e.g., therapeutic peptide) is an antibody or a fragment or modification thereof. In some embodiments, the therapeutic peptide is an enzyme, a ligand, a receptor, a transcription factor, or a fragment or modification thereof. In some embodiments, the therapeutic peptide is an antimicrobial peptide or a fragment or modification thereof.

[0017] In some embodiments, the EVs (e.g., exosomes) further comprise a second scaffolding protein, wherein the second scaffolding protein comprises MARCKS, MARCKSL1, BASP1, or a fragment thereof. In some embodiments, the EVs (e.g., exosomes) further comprise a second scaffolding protein, wherein the second scaffolding protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment thereof.

[0018] In some embodiments, the scaffolding protein comprises a peptide sequence corresponding to the pattern (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118) or the pattern without the N-terminal (M). In some embodiments, the scaffolding protein comprises the peptide (M)(G)(π)(X)(Φ / π)(π)(+)(+) or the peptide without the N-terminal (M), where each position within the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu). In some embodiments, the scaffolding protein comprises the peptide sequence (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+) or the peptide without the N-terminal (M), where each position within the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu). For amino acid nomenclature, see R. Aasland et al., FEBS Letters 513 (2002): 141-144.

[0019] In some embodiments, the scaffold protein comprises a peptide of any one of SEQ ID NO: 4-110. In some embodiments, the scaffold protein comprises the peptide of MGXKLSKKK (SEQ ID NO: 116) or the peptide without the N-terminal M, where X is any amino acid. In some embodiments, the scaffold protein comprises the peptide of SEQ ID NO: 110, or the corresponding peptide without the N-terminal M. In some embodiments, the scaffold protein comprises the peptide of SEQ ID NO: 13, or the corresponding peptide without the N-terminal (M).

[0020] In some embodiments, the scaffold protein further comprises a payload, for example, a peptide.

[0021] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an EV (e.g., exosome) of the present disclosure and an excipient.

[0022] In some embodiments, the pharmaceutical composition is substantially free of macromolecules, where the macromolecules are selected from nucleic acids, exogenous proteins, lipids, carbohydrates, metabolites, and combinations thereof.

[0023] In yet another embodiment, the present disclosure provides a cell population for generating an EV (e.g., exosome) provided herein.

[0024] In some embodiments, the cell population comprises an exogenous sequence encoding a scaffold protein, the scaffold protein comprising MARCKS, MARCKSL1, BASP1, or a fragment or modification thereof. In some embodiments, the cell population further comprises a second exogenous sequence encoding a second scaffold protein, where the second scaffold protein comprises MARCKS, MARCKSL1, BASP1, or a fragment or modification thereof. In some embodiments, the cell population further comprises a second exogenous sequence encoding a second scaffold protein, where the second scaffold protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment thereof.

[0025] In some embodiments, the exogenous sequence is inserted into the genomic sequence encoding MARCKS, MARCKSL1, or BASP1, where the exogenous sequence and the genomic sequence encode a scaffold protein. In some embodiments, the exogenous sequence is in a plasmid.

[0026] In some embodiments, the exogenous sequence encodes a bioactive molecule (e.g., a therapeutic peptide). In some embodiments, the therapeutic peptide is selected from the group consisting of: a natural peptide, a recombinant peptide, a synthetic peptide, or a linker attached to a therapeutic compound. In some embodiments, the therapeutic compound is selected from the group consisting of: a nucleotide, an amino acid, a lipid, a carbohydrate, and a small molecule. In some embodiments, the therapeutic peptide is an antibody or a fragment or a modification thereof. In certain embodiments, the antibody is a nanobody. One of ordinary skill in the art will understand that the antibodies used in the EVs of the present disclosure can be any antigen-binding molecule known in the art, including, for example, alternative antibody forms, antibody-drug conjugates (ADCs), or immunotoxins.

[0027] In some embodiments, the therapeutic peptide is an enzyme, a ligand, a receptor, a transcription factor, or a fragment or a modification thereof. In some embodiments, the therapeutic peptide is an antimicrobial peptide or a fragment or a modification thereof.

[0028] In some embodiments, the exogenous sequence encodes a targeting moiety. In some embodiments, the targeting moiety is specific for an organ, a tissue, or a cell.

[0029] In some embodiments, the second scaffold protein further comprises a targeting moiety. In some embodiments, the targeting moiety is specific for an organ, a tissue, or a cell.

[0030] In one embodiment, the present disclosure provides a polypeptide for modifying EVs (e.g., exosomes), the polypeptide comprising the following sequences:

[0031] (i) (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO:118) or the corresponding sequence without the N-terminal (M);

[0032] (ii) (M)(G)(π)(X)(Φ / π)(π)(+)(+) or the corresponding sequence without the N-terminal (M), where each position in the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu); or

[0033] (iii)(M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+) or a corresponding sequence lacking the N-terminal (M), where each position inside the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu).

[0034] In some embodiments, the polypeptide of the scaffold comprises the sequence of any one of SEQ ID NOs: 4-110, or any corresponding sequence lacking the N-terminal M. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 13 or a corresponding sequence lacking the N-terminal M. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 110 or a corresponding sequence lacking the N-terminal M. In some embodiments, the polypeptide comprises the sequence MGXKLSKKK (SEQ ID NO: 116), where X is any amino acid, or a corresponding sequence lacking the N-terminal M.

[0035] In one aspect, the present disclosure provides a polynucleotide construct comprising a coding sequence encoding a polypeptide provided herein. In some embodiments, the coding sequence is codon-optimized.

[0036] In another aspect, the present disclosure provides a method for preparing engineered EVs (e.g., exosomes), which comprises the following steps:

[0037] a. Introducing a nucleic acid construct encoding a fusion polypeptide into a cell, the fusion polypeptide comprising

[0038] (i) a first sequence encoding MARCKS, MARCKSL1, BASP1 or a fragment or a modification thereof, and

[0039] (ii) a second sequence encoding a payload (e.g., a bioactive molecule such as a therapeutic peptide);

[0040] b. Maintaining the cell under conditions that permit the cell to express the fusion polypeptide; and,

[0041] c. Obtaining engineered exosomes comprising the fusion polypeptide from the cell.

[0042] In some embodiments, the first sequence comprises the following sequence;

[0043] (i) (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118) or a corresponding sequence lacking the N-terminal (M);

[0044] (ii) (M)(G)(π)(X)(Φ / π)(π)(+)(+) or a corresponding sequence lacking the N-terminal (M), wherein each position within the parentheses represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu); or

[0045] (iii) (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+) or a corresponding sequence lacking the N-terminal (M), wherein each position within the parentheses represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu).

[0046] In some embodiments, the polynucleotide comprises the sequence of any one of SEQ ID NOs: 4 - 110, or any corresponding sequence lacking the N-terminal M. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 13 or a corresponding sequence lacking the N-terminal (M). In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 110 or a corresponding sequence lacking the N-terminal (M). In some embodiments, the polynucleotide comprises the sequence MGXKLSKKK (SEQ ID NO: 116) or a corresponding sequence lacking the N-terminal M, wherein X is any amino acid.

[0047] In some embodiments, the fusion polypeptide is present on the luminal surface of engineered EVs (e.g., engineered exosomes) at a higher density than different scaffold proteins in different EVs (e.g., exosomes), wherein the different scaffold proteins include conventional EV (e.g., exosome) proteins or variants thereof. In some embodiments, the fusion polypeptide is present at a density that is more than 2-fold that of different scaffold proteins in different EVs (e.g., exosomes).

[0048] In some embodiments, the fusion polypeptide is present at a density that is more than 4-fold, 16-fold, 100-fold, or 10,000-fold that of different scaffold proteins in different EVs (e.g., exosomes).

[0049] The present disclosure relates to isolated extracellular vesicles (EVs) that comprise a bioactive molecule linked to a scaffold protein, wherein the scaffold protein comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND and ED associate with the luminal surface of the EV through ionic interactions, and wherein the ED comprises at least two consecutive lysines (Lys) in the sequence. In some embodiments, the ND associates with the luminal surface of the EV through myristoylation. In other embodiments, the ND has Gly at the N-terminus.

[0050] In some embodiments, the ED comprises at least three Lys, at least four Lys, at least five Lys, at least six Lys, or at least seven Lys. In other embodiments, the ED is linked to the ND by a peptide bond. In some embodiments, the ED comprises (Lys)n, where n is an integer between 1 and 10. In other embodiments, the ED comprises KK, KKK, KKKK (SEQ ID NO:151), KKKKK (SEQ ID NO:152), or any combination thereof.

[0051] In other embodiments, the ND comprises an amino acid sequence as shown in G:X2:X3:X4:X5:X6, wherein G is glycine represented as Gly; wherein ":" represents a peptide bond, wherein each of X2 to X6 is independently an amino acid; and wherein X6 comprises a basic amino acid. In some embodiments, X6 is selected from the group consisting of Lys, Arg, and His.

[0052] In other embodiments, the present disclosure is isolated extracellular vesicles (EVs) that comprise a bioactive molecule linked to a scaffold protein, wherein the scaffold protein comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND comprises an amino acid sequence as shown in G:X2:X3:X4:X5:X6, wherein G is glycine represented by Gly; wherein ":" represents a peptide bond; wherein each of X2 to X6 is independently an amino acid; wherein X6 comprises a basic amino acid; and wherein the ED is linked to X6 by a peptide bond and comprises at least one lysine at the N-terminus of the ED. In some embodiments, the ED does not comprise a transmembrane domain or a cytoplasmic domain of a virus. In some embodiments, X2 is selected from the group consisting of Pro, Gly, Ala, and Ser. In other embodiments, X4 is selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Gln, and Met. In some embodiments, X5 is selected from the group consisting of Pro, Gly, Ala, and Ser.

[0053] In some embodiments, the ND of the scaffold protein comprises the amino acid sequence G:X2:X3:X4:X5:X6, wherein

[0054] G represents Gly;

[0055] ":" represents a peptide bond;

[0056] X2 is an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser;

[0057] X3 is an amino acid;

[0058] X4 is an amino acid selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Gln, and Met;

[0059] X5 is an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser; and

[0060] X6 is an amino acid selected from the group consisting of Lys, Arg, and His.

[0061] In some embodiments, X3 is selected from the group consisting of Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, and Arg.

[0062] In some embodiments, the ND and the ED are linked by a linker. In some embodiments, the linker comprises one or more amino acids.

[0063] In some embodiments, the present disclosure relates to isolated extracellular vesicles (EVs) that comprise a bioactive molecule linked to a scaffold protein, wherein the scaffold protein comprises ND-ED, wherein:

[0064] ND comprises G:X2:X3:X4:X5:X6; wherein:

[0065] G represents Gly;

[0066] “:” represents a peptide bond;

[0067] X2 is an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser;

[0068] X3 is an amino acid;

[0069] X4 is an amino acid selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Glu, and Met;

[0070] X5 is an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser;

[0071] X6 is an amino acid selected from the group consisting of Lys, Arg, and His;

[0072] “—” is an optional linker comprising one or more amino acids; and

[0073] ED is an effector domain that comprises (i) at least two consecutive lysines (Lys) that are linked to X6 by a peptide bond or one or more amino acids; or (ii) at least one lysine that is directly linked to X6 by a peptide bond.

[0074] In some embodiments, X2 is selected from the group consisting of Gly and Ala. In some embodiments, X3 is Lys. In some embodiments, X4 is Leu or Glu. In some embodiments, X5 is selected from the group consisting of Ser and Ala. In some embodiments, X6 is Lys. In other embodiments, X2 is Gly, Ala, or Ser; X3 is Lys or Glu, X4 is Leu, Phe, Ser, and Glu, X5 is Ser or Ala; and X6 is Lys.

[0075] In some embodiments, ND and ED are linked by a linker comprising one or more amino acids. In some embodiments, ED comprises Lys (K), KK, KKK, KKKK (SEQ ID NO:151), KKKKK (SEQ ID NO:152), or any combination thereof.

[0076] In some embodiments, the scaffold protein comprises an amino acid sequence selected from the group consisting of: (i) GGKLSKK (SEQ ID NO:157), (ii) GAKLSKK (SEQ ID NO:158), (iii) GGKQSKK (SEQ ID NO:159), (iv) GGKLAKK (SEQ ID NO:160), or (v) any combination thereof. In some embodiments, the scaffold protein comprises an amino acid sequence selected from the group consisting of: (i) GGKLSKKK (SEQ ID NO:161), (ii) GGKLSKKS (SEQ ID NO:162), (iii) GAKLSKKK (SEQ ID NO:163), (iv) GAKLSKKS (SEQ ID NO:164), (v) GGKQSKKK (SEQ ID NO:165), (vi) GGKQSKKS (SEQ ID NO:166), (vii) GGKLAKKK (SEQ ID NO:167), (viii) GGKLAKKS (SEQ ID NO:168), and (ix) any combination thereof. In some embodiments, the scaffold protein has a length of at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 105, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 amino acids.

[0077] In some embodiments, the scaffold protein comprises (i) GGKLSKKKKGYNVN (SEQ ID NO:169), (ii) GAKLSKKKKGYNVN (SEQ ID NO:170), (iii) GGKQSKKKKGYNVN (SEQ ID NO:171), (iv) GGKLAKKKKGYNVN (SEQ ID NO:172), (v) GGKLSKKKKGYSGG (SEQ ID NO:173), (vi) GGKLSKKKKGSGGS (SEQ ID NO:174), (vii) GGKLSKKKKSGGSG (SEQ ID NO:175), (viii) GGKLSKKKSGGSGG (SEQ ID NO:176), (ix) GGKLSKKSGGSGGS (SEQ ID NO:177), (x) GGKLSKSGGSGGSV (SEQ ID NO:178), or (xi) GAKKSKKRFSFKKS (SEQ ID NO:179).

[0078] In some embodiments, the scaffold protein does not contain a Met at the N-terminus. In some embodiments, the scaffold protein comprises a myristoylated amino acid residue at the N-terminus of the scaffold protein. In some embodiments, the amino acid residue at the N-terminus of the scaffold protein is Gly. In some embodiments, the amino acid residue at the N-terminus of the scaffold protein is synthetic. In some embodiments, the amino acid residue at the N-terminus of the scaffold protein is a glycine analogue.

[0079] In some embodiments, the scaffold protein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO:1 (MARKS), SEQ ID NO:2 (MARCKSL1), or SEQ ID NO:3 (BASP1).

[0080] In some embodiments, the bioactive molecule is on the luminal surface or within the lumen of the EV. In some embodiments, the scaffold protein further comprises a transmembrane domain. In some embodiments, the transmembrane domain is between the ED domain of the scaffold protein and the bioactive molecule. In some embodiments, the scaffold protein further comprises a vesicle exterior domain. In some embodiments, the bioactive molecule is linked to the vesicle exterior domain.

[0081] In some embodiments, the scaffolding protein is linked to the bioactive molecule via a linker. In some embodiments, the ND domain is linked to the ED domain via a linker. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker comprises a cleavable linker. In some embodiments, the linker comprises a flexible linker.

[0082] In other embodiments, the bioactive molecule comprises a protein, polypeptide, peptide, polynucleotide (DNA and / or RNA), compound, virus, ionophore, a carrier of the ionophore, a moiety forming a channel or pore, or any combination thereof. In some embodiments, the protein comprises a recombinant peptide, natural peptide, synthetic peptide, antibody, fusion protein, or any combination thereof. In some embodiments, the protein comprises an enzyme, cytokine, ligand, receptor, transcription factor, or a combination thereof. In some embodiments, the virus comprises an adeno-associated virus, parvovirus, retrovirus, adenovirus, or any combination thereof.

[0083] In other embodiments, the EV further comprises a second scaffolding protein. In some embodiments, the second scaffolding protein comprises a PTGFRN polypeptide, BSG polypeptide, IGSF2 polypeptide, IGSF3 polypeptide, IGSF8 polypeptide, ITGB1 polypeptide, ITGA4 polypeptide, SLC3A2 polypeptide, ATP transporter polypeptide, aminopeptidase N (ANPEP) polypeptide, ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1) polypeptide, neprilysin (MME) polypeptide, neuropilin-1 (NRP1) polypeptide, or a fragment thereof. In some embodiments, the bioactive molecule is an inhibitor of a negative checkpoint modulator or an inhibitor of a binding partner of a negative checkpoint modulator. In some embodiments, the negative checkpoint modulator is selected from the group consisting of cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell activation V domain Ig inhibitor (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73. In some embodiments, the bioactive molecule is an immunogenic protein.

[0084] In other embodiments, the bioactive molecule is a toxin, a toxoid, or a non-toxic mutant of a toxin. In some embodiments, the toxin is diphtheria toxin. In some embodiments, the toxoid is tetanus toxoid. In some embodiments, the bioactive molecule is a non-toxic mutant of diphtheria toxin.

[0085] In some embodiments, the bioactive molecule is an activator of a co-stimulatory molecule or an activator of a binding partner of a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is a member of the TNF receptor superfamily. In some embodiments, the members of the TNF receptor superfamily are selected from the group consisting of: CD120a, CD120b, CD18, OX40, CD40, Fas receptor, M68, CD27, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, RANK, OCIF, TWEAK receptor, TACI, BAFF receptor, ATAR, CD271, CD269, AITR, TROY, CD358, TRAMP, and XEDAR. In some embodiments, the activator of the co-stimulatory molecule is a member of the TNF superfamily. In some embodiments, the members of the TNF superfamily are selected from the group consisting of: TNFα, TNF-C, OX40L, CD40L, FasL, LIGHT, TL1A, CD27L, Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand, and EDA-2. In some embodiments, the co-stimulatory molecule is a CD28 superfamily co-stimulatory molecule. In some embodiments, the CD28 superfamily co-stimulatory molecule is ICOS or CD28. In some embodiments, the activator of the co-stimulatory molecule is ICOSL, CD80, or CD86.

[0086] In other embodiments, the cytokine is selected from the group consisting of: IL-2, IL-7, IL-10, IL-12, and IL-15. In some embodiments, the protein comprises a T cell receptor (TCR), a T cell co-receptor, a major histocompatibility complex (MHC), a human leukocyte antigen (HLA), or a derivative thereof. In some embodiments, the protein comprises a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, alpha-folate receptor, CE7R, IL-3, cancer-testis antigen, MART-1 gp100, and tumor necrosis factor-related apoptosis-inducing ligand.

[0087] In other embodiments, the EV is an exosome.

[0088] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising the EVs of the present disclosure and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure relates to a cell that produces the EVs of the present disclosure. In some embodiments, the present disclosure relates to a kit comprising the EVs of the present disclosure and instructions for use.

[0089] In other embodiments, the present invention relates to a method for preparing EVs, the method comprising culturing the cells of the present disclosure under suitable conditions and obtaining EVs. In some embodiments, the present disclosure relates to a method for anchoring a bioactive molecule to an extracellular vesicle, which comprises linking the bioactive molecule to a scaffold protein disclosed herein.

[0090] In other embodiments, the present disclosure relates to a method for preventing or treating a disease in a subject in need thereof, which comprises administering the EVs of the present disclosure, wherein the disease is related to an antigen. In some embodiments, the EVs are administered parenterally, orally, intravenously, intramuscularly, intratumorally, intranasally, subcutaneously, or intraperitoneally.

[0091] Embodiments

[0092] E1. EVs (e.g., exosomes) comprising a scaffold protein, wherein at least a portion of the scaffold protein is expressed by an exogenous sequence, and the scaffold protein comprises MARCKS, MARCKSL1, BASP1, or a fragment or modification thereof.

[0093] E2. An EV (e.g., exosome) of embodiment E1, wherein the scaffolding protein is present in the EV (e.g., exosome) at a higher density compared to different scaffolding proteins in different EVs (e.g., exosomes), and wherein the different scaffolding proteins include conventional EV (e.g., exosome) proteins or variants thereof.

[0094] E3. An EV (e.g., exosome) of embodiment E2, wherein the conventional EV (e.g., exosome) protein is selected from the group consisting of: CD9, CD63, CD81, PDGFR, GPI-anchored protein, lactadherin, LAMP2, LAMP2B, and fragments thereof.

[0095] E4. An EV (e.g., exosome) of any one of embodiments E1 to E3, wherein the EV (e.g., exosome) is produced by a cell genetically modified to contain an exogenous sequence, optionally wherein the cell is a HEK293 cell.

[0096] E5. An EV (e.g., exosome) of embodiments E1 to E4, wherein the cell contains a plasmid containing the exogenous sequence.

[0097] E6. An EV (e.g., exosome) of embodiments E1 to E5, wherein the cell contains an exogenous sequence inserted into the cell genome.

[0098] E7. An EV (e.g., exosome) of embodiments E1 to E6, wherein the exogenous sequence is inserted into a genomic locus that is 3' or 5' relative to the genomic sequence encoding MARCKS, MARCKSL1, or BASP1.

[0099] E8. An EV (e.g., exosome) of embodiments E1 to E7, wherein the exogenous sequence is inserted into the genomic sequence encoding MARCKS, MARCKSL1, or BASP1.

[0100] E9. An EV (e.g., exosome) according to any one of embodiments E1 to E8, wherein the scaffolding protein is a fusion protein comprising MARCKS, MARCKSL1, or BASP1 or a fragment thereof and a therapeutic peptide.

[0101] E10. An EV (e.g., exosome) of embodiment E9, wherein the therapeutic peptide is selected from the group consisting of: natural peptides, recombinant peptides, synthetic peptides, or a linker attached to a therapeutic compound.

[0102] E11. An EV (e.g., exosome) of embodiment E9, wherein the therapeutic compound is selected from the group consisting of: nucleotides, amino acids, lipids, carbohydrates, and small molecules.

[0103] EV (e.g., exosome) of embodiment E9, wherein the therapeutic peptide is an antibody or a fragment or modification thereof.

[0104] EV (e.g., exosome) of embodiment E9, wherein the therapeutic peptide is an enzyme, ligand, receptor, transcription factor or a fragment or modification thereof.

[0105] EV (e.g., exosome) of embodiment E9, wherein the therapeutic peptide is an antimicrobial peptide or a fragment or modification thereof.

[0106] EV (e.g., exosome) according to any one of embodiments E1 to E14, wherein the EV further comprises a second scaffold protein, and the second scaffold protein comprises MARCKS, MARCKSL1, BASP1 or a fragment thereof.

[0107] EV (e.g., exosome) according to any one of embodiments E1 to E14, wherein the EV further comprises a second scaffold protein, and the second scaffold protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter or a fragment thereof.

[0108] EV (e.g., exosome) according to any one of embodiments E1 to E16, wherein the scaffold protein comprises a peptide of the sequence (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118), or the corresponding sequence without the N-terminal (M).

[0109] EV (e.g., exosome) according to any one of embodiments E1 to E17, wherein the scaffold protein comprises a peptide of the sequence (M)(G)(π)(X)(Φ / π)(π)(+)(+) or the corresponding sequence without the N-terminal (M), where each position in the parentheses represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu).

[0110] An EV (e.g., exosome) according to any one of embodiments E1 to E18, wherein the scaffold protein comprises a peptide of the sequence (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), or the corresponding sequence without the N-terminal (M), where each position in parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu).

[0111] E20. An EV (e.g., exosome) according to any one of embodiments E18 or E19, wherein the scaffold protein comprises a peptide of any one of SEQ ID NO: 4 - 110, or any corresponding sequence without the N-terminal M.

[0112] E21. An EV (e.g., exosome) according to any one of embodiments E18 or E19, wherein the scaffold protein comprises the peptide MGXKLSKKK (SEQ ID NO: 116), or the corresponding sequence without the N-terminal M, where X is any amino acid.

[0113] E22. An EV (e.g., exosome) according to embodiment E20, wherein the scaffold protein comprises the peptide of SEQ ID NO: 110, or the corresponding sequence without the N-terminal M.

[0114] E23. An EV (e.g., exosome) according to embodiment E20, wherein the scaffold protein comprises the peptide of SEQ ID NO: 13 or the corresponding sequence without the N-terminal M.

[0115] E24. An EV (e.g., exosome) according to any one of embodiments E1 to E24, wherein the scaffold protein further comprises a payload, e.g., a bioactive molecule such as a peptide.

[0116] E25. A pharmaceutical composition comprising an EV (e.g., exosome) according to any one of embodiments E1 to E24 and an excipient.

[0117] E26. The pharmaceutical composition of embodiment E25, which is substantially free of macromolecules, wherein the macromolecules are selected from nucleic acids, exogenous proteins, lipids, carbohydrates, metabolites, and combinations thereof.

[0118] E27. A cell population for producing EVs (e.g., exosomes) according to any one of embodiments E1 to E24.

[0119] E28. The cell population of embodiment E27, which comprises an exogenous sequence encoding a scaffolding protein, said scaffolding protein comprising MARCKS, MARCKSL1, BASP1 or a fragment or modification thereof.

[0120] E29. The cell population of embodiment E28, said cell population further comprising a second exogenous sequence encoding a second scaffolding protein, wherein said second scaffolding protein comprises MARCKS, MARCKSL1, BASP1 or a fragment or modification thereof.

[0121] E30. The cell population of embodiment E28, said cell population further comprising a second exogenous sequence encoding a second scaffolding protein, wherein said second scaffolding protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter or a fragment thereof.

[0122] E31. The cell population according to any one of embodiments E27 to E30, wherein the exogenous sequence is inserted into a genomic sequence encoding MARCKS, MARCKSL1 or BASP1, and wherein the exogenous sequence and the genomic sequence encode a scaffolding protein.

[0123] E32. The cell population according to any one of embodiments E27 to E30, wherein the exogenous sequence is in a plasmid.

[0124] E33. The cell population according to any one of embodiments E27 to E32, wherein the exogenous sequence encodes a bioactive molecule (e.g., a therapeutic peptide).

[0125] E34. The cell population of embodiment E33, wherein the therapeutic peptide is selected from the group consisting of: a natural peptide, a recombinant peptide, a synthetic peptide or a linker attached to a therapeutic compound.

[0126] E35. The cell population of embodiment E33, wherein the therapeutic compound is selected from the group consisting of: nucleotides, amino acids, lipids, carbohydrates and small molecules.

[0127] E36. The cell population of embodiment E33, wherein the therapeutic peptide is an antibody or a fragment or modification thereof.

[0128] E37. The cell population of embodiment E33, wherein the therapeutic peptide is an enzyme, a ligand, a receptor, a transcription factor or a fragment or modification thereof.

[0129] The cell population of embodiment E33, wherein the therapeutic peptide is an antimicrobial peptide or a fragment or modification thereof.

[0130] The cell population of embodiment E28, wherein the exogenous sequence encodes a targeting moiety.

[0131] The cell population of embodiment E39, wherein the targeting moiety is specific for an organ, tissue, or cell.

[0132] The cell population of embodiment E29 or E30, wherein the second scaffold protein further comprises a targeting moiety.

[0133] The cell population of embodiment E41, wherein the targeting moiety is specific for an organ, tissue, or cell.

[0134] A polypeptide for modifying EVs (e.g., exosomes), comprising the following sequences:

[0135] (i) (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO:118) or the corresponding sequence without the N-terminal (M);

[0136] (ii) (M)(G)(π)(X)(Φ / π)(π)(+)(+) or the corresponding sequence without the N-terminal (M), where each position inside the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu); or

[0137] (iii) (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+) or the corresponding sequence without the N-terminal (M), where each position inside the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu).

[0138] The polypeptide of embodiment E43, which comprises any one of the sequences of SEQ ID NO: 4-110, or any corresponding sequence without N-terminal M.

[0139] The polypeptide of embodiment E43, which comprises the sequence of SEQ ID NO: 13 or the corresponding sequence without N-terminal M.

[0140] The polypeptide of embodiment E43, which comprises the sequence of SEQ ID NO: 110 or the corresponding sequence without N-terminal M.

[0141] The polypeptide of embodiment E43, which comprises the sequence MGXKLSKKK (SEQ ID NO: 116) or the corresponding sequence without N-terminal M, wherein X is any amino acid.

[0142] The polypeptide according to any one of embodiments E43 to E47, wherein the polypeptide is fused with a payload (such as a bioactive molecule such as a peptide).

[0143] The polypeptide of embodiment E48, wherein the polypeptide is fused to the N-terminus of the peptide.

[0144] A polynucleotide construct comprising a coding sequence encoding the polypeptide according to any one of embodiments E43 to E49.

[0145] The polynucleotide construct of embodiment E50, wherein the coding sequence is codon-optimized.

[0146] A method for preparing engineered EVs (e.g., exosomes), the method comprising the steps of:

[0147] a. introducing a nucleic acid construct encoding a fusion polypeptide into a cell, the nucleic acid construct comprising (i) a first sequence encoding MARCKS, MARCKSL1, BASP1 or a fragment or modification thereof, and (ii) a second sequence encoding a payload, such as a bioactive molecule such as a peptide;

[0148] b. maintaining the cell under conditions that allow the cell to express the fusion polypeptide; and,

[0149] c. obtaining engineered EVs (e.g., exosomes) containing the fusion polypeptide from the cell.

[0150] The method of embodiment E52, wherein the first sequence comprises the following sequence:

[0151] (i) (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118) or a corresponding sequence lacking the N-terminal (M);

[0152] (ii) (M)(G)(π)(X)(Φ / π)(π)(+)(+) or a corresponding sequence lacking the N-terminal (M), where each position within the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu); or

[0153] (iii) (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+) or a corresponding sequence lacking the N-terminal (M), where each position within the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu).

[0154] E54. The method according to any one of embodiments E52 or E53, wherein the first sequence comprises any one of SEQ ID NOs: 4 - 110, or any corresponding sequence lacking the N-terminal M.

[0155] E55. The method of embodiment E54, wherein the first sequence comprises SEQ ID NO: 13, or a corresponding sequence lacking the N-terminal M.

[0156] E56. The method of embodiment E55, wherein the first sequence comprises SEQ ID NO: 110, or a corresponding sequence lacking the N-terminal M.

[0157] E57. The method of embodiment E53, wherein the first sequence comprises MGXKLSKKK (SEQ ID NO: 116) or a corresponding sequence lacking the N-terminal M, where X is any amino acid.

[0158] The method according to any one of embodiments E52 to E57, wherein the fusion polypeptide is present on the luminal surface of the engineered EV (e.g., exosome) at a higher density than different scaffold proteins in different EVs (e.g., exosomes), wherein the different scaffold proteins include conventional EVs, such as exosomal proteins or variants thereof.

[0159] E59. The method of embodiment E58, wherein the fusion polypeptide is present at a density that is more than 2-fold that of different scaffold proteins in different EVs (e.g., exosomes).

[0160] E60. The method of embodiment E59, wherein the fusion polypeptide is present at a density that is more than 4-fold, 16-fold, 100-fold, or 10,000-fold that of different scaffold proteins in different EVs (e.g., exosomes). BRIEF DESCRIPTION OF THE DRAWINGS

[0161] The drawings depict various aspects of the present disclosure for illustrative purposes only. Those skilled in the art will readily recognize from the following discussion that alternative aspects of the structures and methods shown herein may be employed without departing from the principles of the present disclosure described herein.

[0162] Figure 1A Showing the sequences of the fusion proteins (SEQ ID NO: 135 - 142, in the order of appearance), the fusion proteins comprising BASP1 fragments fused to tags and GFP. Figure 1B Showing the results of anti- Figure 1A protein blotting of exosomes purified from cells stably expressing one of the fusion proteins in Figure 1A and Figure 1B For

[0163] Figure 2A Showing the sequences of the BASP1 fragment (1 - 30) (SEQ ID NO: 4) fused to tags and GFP and its modifications (1 - 30 - S6D, 1 - 30 - S6A, and 1 - 30 - L5Q) (SEQ ID NO: 143 - 145 in the order of appearance).

[0164] Figure 2B Showing the results of anti- Figure 2A protein blotting of exosomes purified from cells stably expressing one of the fusion proteins in Figure 2A and 2B, amino acid positions, e.g., BASP1 1-30 refers to the BASP-1 fragment initially encoded by the gene construct. The first Met was excised during processing.

[0165] Figure 3 Shows Coomassie stained image of a protein gel of exosome samples purified from cells that stably express full-length MARCKSL1, BASP1, or MARCKS, MARCKSL1 or BASP1 amino acids 1-30 all fused to -GFP. The white arrows on the image indicate the bands corresponding to the fusion proteins. For Figure 3 , amino acid positions, e.g., BASP1, 1-30 refers to the BASP-1 fragment initially encoded by the gene construct. The first Met was excised during processing.

[0166] Figure 4 Shows a protein sequence alignment between the first 28 amino acids of BASP1 (conserved region 1), amino acids 1-7 and 152-173 of MARCKS (conserved region 2), and amino acids 1-7 and 87-110 of MARCKSL1 (conserved region 3).

[0167] Figure 5A Shows the sequences of BASP1 amino acids 1-30 ("BASP1-30") (SEQ ID NO:4) and fusion proteins (SEQ ID NOS:146-149 in the order of appearance), the fusion proteins comprising amino acids 1-3 of MARCKS or a modification thereof fused to the PSD domain of MARCKS or a modification thereof ("MARCKS-MG-PSD", "MARCKS-MA-PSD", "MARCKS-MG-PSD-K6S", and "MARCKS-MG-PSD-K6A"). Point mutations introduced into the MARCKS sequence are shown in bold. Amino acid positions, e.g., aa 1-30 of BASP1 or aa 1-3 of MARCKS, refer to the fragments initially encoded by the gene construct. The first Met was excised during processing.

[0168] Figure 5B Shows the results of an anti western blot of exosomes purified from cells that stably express a fusion protein comprising Figure 5A amino acid sequences of

[0169] Figure 6Shows three different consensus sequences (all three sequences are represented by SEQ ID NO:118) derived from functional studies of MARCKS, MARCKSL1, and BASP1, and the amino acid requirements for each of the sequences used to load a payload into the lumen of an exosome by attachment to the luminal surface.

[0170] Figure 7A Shows total protein (upper panel) and anti-Cas9 western blot (lower panel) of native exosomes or exosomes purified from cells and decreasing amounts of recombinant Cas9, where the cells stably express Cas9 fused to amino acids 1-10 or 1-30 of BASP1. Amino acid positions, e.g., aa 1-10 of BASP or aa 1-30 of BASP1, refer to the fragment initially encoded by the gene construct. The first Met was excised during processing.

[0171] Figure 7B The upper panel shows the Figure 7A standard curve of Cas9 densitometry of the western blot results. The lower panel also provides the amount of loaded Cas9 in each purified exosome estimated based on the standard curve, where the Cas9 is in the form of a fusion protein conjugated to a fragment of 1-30 amino acids or 1-10 amino acids of BASP1 (2-30 amino acids or 2-10 amino acids of BASP1 after processing (i.e., the first Met is cut off)).

[0172] Figure 8A Shows a protein gel image of exosomes purified from cells that were stably transfected with a construct expressing the N-terminus of BASP1 (amino acids 1-10) fused to ovalbumin ("BASP1(1-10)-OVA"), or cells stably transfected with two constructs (one expressing the N-terminus of BASP1 (amino acids 1-10) fused to ovalbumin and the other expressing CD40L fused to the transmembrane protein PTGFRN ("BASP1(1-10)-OVA; 3XCD40L-PTGFRN")). Figure 8A Also shows an image of a protein gel loaded with decreasing amounts of recombinant OVA. Amino acid numbering, e.g., amino acids 1-10, refers to the fragment initially encoded by the gene construct. The first Met was excised during processing.

[0173] Figure 8B Shows the Figure 8A anti-ovalbumin western blot results of samples from

[0174] Figure 8C Shows western blot results comparing recombinant ova and ova fused to exoTOPE. Figure 8DShows a schematic diagram of exosomes comprising (i) an exoTOPE linked to ovalbumin on the luminal side of the exosome and (ii) an immunostimulant in the lumen of the exosome.

[0175] Figure 9A Shows the sequence (SEQ ID NO:150) of a camelid nanobody against GFP fused to amino acids 1 - 10 of BASP1 and a tag. Amino acid positions, e.g., amino acids 1 - 10 of BASP1, refer to the fragment initially encoded by the gene construct. The first Met is excised during processing.

[0176] Figure 9B Shows a protein gel of exosomes purified from cells and an anti - protein blot result of cells stably expressing Figure 10 a fusion protein of A ("BASP1(1 - 10)-nanobody") or a protein lacking the BASP1 sequence ("nanobody"). Amino acid numbering, e.g., BASP1(1 - 10)-nanobody, refers to the fragment initially encoded by the gene construct. The first Met is excised during processing.

[0177] Figure 10 Shows a schematic diagram of an exosome mRNA loading system comprising (i) BASP1(1 - 30) fused to and monomeric or dimeric MCP variants (1XMCP(V29I) ("815"; SEQ ID NO:111), 1XMCP(V29I / N55K) ("817"; SEQ ID NO:112), 2XMCP(V29I) ("819"; SEQ ID NO:113) or 2XMCP(V29I / N55K)) ("821"; SEQ ID NO:114)) and (ii) a luciferase mRNA containing 3x MS2 hairpin loops ("luciferase - MS2 mRNA" or "811"; SEQ ID NO:115). Amino acid numbering, e.g., BASP1(1 - 30), refers to the fragment initially encoded by the gene construct. The first Met is excised during processing.

[0178] Figure 11A Shows a protein gel of exosomes comprising Figure 10 the mRNA loading constructs described in (combinations of luciferase mRNA (811) with various BASP1 fusion proteins (815, 817 or 819)).

[0179] Figure 11B Shows Figure 11A the anti - Western blot.

[0180] Figure 12A Shows RT-qPCR results of the amount of luciferase mRNA in cells (top) or exosomes (bottom) containing the Figure 10 shown mRNA-loading construct.

[0181] Figure 12B Shows a table quantifying the amount of luciferase mRNA (including the enrichment fold from random loading of luciferase mRNA) in purified exosomes from samples in Figure 12A .

[0182] Figure 13 Shows a schematic of the CD40L trimer with extracellular surface display of a transmembrane protein fused to the N-terminal fragments of MARCKS, MARCKSL1, and BASP1 to allow anchoring to the luminal surface of exosomes. Amino acid numbers, e.g., MARCKS 1-30, MARCKSL1 1-30, BASP1 1-30, BASP1 1-10ExoTOPE, refer to the fragments initially encoded by the gene construct. The first Met was excised during processing.

[0183] Figure 14A Shows the results of mouse B cell activation in cultures incubated with exosomes with surface expression of CD40L fused to the N-terminal fragments of MARCKS, MARCKSL1, and BASP1.

[0184] Figure 14B Shows the results of human B cell activation in cultures incubated with exosomes with surface expression of CD40L fused to the N-terminal fragments of MARCKS, MARCKSL1, and BASP1.

[0185] Figure 14C Shows a graph of the relative potency of different CD40L surface-displaying exosomes when fused to the N-terminal sequences or full-length PTGFRN of MARCKS, MARCKSL1, and BASP1. Amino acid numbers, e.g., BASP1 1-30, refer to the fragments initially encoded by the gene construct. The first Met was excised during processing.

[0186] Figure 15 Shows the number of peptide spectral matches (PSMs) of luminal proteins (MARCKS, MARCKSL1, and BASP1) and conventional EV (e.g., exosome) proteins (CD81 and CD9) in exosomes purified from various cell lines from different sources (HEK293SF, kidney; HT1080, connective tissue; K562, bone marrow; MDA-MB-231, breast; Raji, lymphoblast; mesenchymal stem cells (MSC), bone marrow).

[0187] Figure 16 Exosomes derived from Chinese hamster ovary (CHO) cells alone or from cells overexpressing Protein gel of exosomes from cells expressing BASP1 or BASP1 N-terminal fragments (1-30 or 1-8) fused to BASP1 or BASP1 fused to GFP (left) and antibodies Western blot (right). Amino acid numbering, e.g., BASP1(1-30), refers to the fragment originally encoded by the gene construct. The first Met is removed during processing. DETAILED DESCRIPTION

[0188] The present disclosure relates to an extracellular vesicle (EV) (e.g., an exosome), comprising at least one bioactive molecule connected to the EV (e.g., an exosome) via a scaffold protein, wherein the scaffold protein comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND and / or the ED associate with the luminal surface of the EV (e.g., an exosome), wherein the ED comprises (i) a lysine repeat sequence in the ED or (ii) a lysine repeat sequence when bound to the ND, e.g., K at the C-terminus in the ND and K at the N-terminus in the ED, wherein the ND is directly connected to the ED, i.e., connected by a peptide bond. The present disclosure also provides a minimum number of amino acids capable of anchoring a payload (e.g., a bioactive molecule) to the luminal surface of an EV (e.g., an exosome), e.g., a fragment of 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, or 7 to 8 amino acids. NDs can be associated with the luminal surface of EVs (e.g., exosomes) through myristoylation, while EDs can be associated with the luminal surface of EVs (e.g., exosomes) through ionic interactions, such as through attractive electrostatic interactions. Non-limiting examples of various aspects are shown in the present disclosure.

[0189] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific compositions or process steps described, and therefore may of course vary. It will be apparent to those skilled in the art upon reading the present disclosure that each individual aspect described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any additional several aspects without departing from the scope or spirit of the present disclosure. Any stated method can be performed in the order of stated events or in any other order that is logically possible.

[0190] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It should also be understood that the terminology used herein is only for the purpose of describing specific aspects and is not intended to be limiting, as the scope of the disclosure will only be limited by the appended claims.

[0191] Accordingly, the following terms, as defined immediately below, are more fully defined by reference to the entire specification.

[0192] I. Definitions

[0193] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide a general dictionary of many of the terms used in this disclosure. As used herein, the following terms have the meanings given to them below.

[0194] It should be noted that the term "a / an" entity refers to one or more of the said entities; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Accordingly, the terms "a / an", "one or more" and "at least one" are used interchangeably herein. It should also be noted that the claims may be drafted to exclude any optional elements. Accordingly, for exclusive terms such as "solely", "only" and the like used in conjunction with the recitation of claim elements, or "negative" limitations used, this specification is only intended to serve as a precedent.

[0195] In addition, when used herein, "and / or" shall be regarded as specifically disclosing each of the two specified features or components, with or without the other. Accordingly, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0196] It should be understood that whenever an aspect is described herein in the language "comprising", additional similar aspects are also provided that are described in the language "consisting of" and / or "consisting essentially of".

[0197] Units, prefixes, and symbols are expressed in their accepted International System of Units (SI) form.

[0198] Numeric ranges include the values defining the range. In the case of listing a range of values, it should be understood that each intervening integer value and each fraction between the listed upper and lower limits of the range are also specifically disclosed, as well as each sub-range between such values. The upper and lower limits of any range can be independently included in or excluded from the range, and each range that includes either limit, excludes both limits, or includes both limits is also covered by the present disclosure.

[0199] Accordingly, the ranges recited herein should be understood as shorthand for all values within the range, including the recited endpoints. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or sub-range selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0200] Unless otherwise indicated, a reference to a compound having one or more stereocenters is intended to refer to each stereoisomer and all combinations of its stereoisomers.

[0201] In the case of explicitly listing values, it should be understood that values that are approximately the same amount or quantity as the recited values are also within the scope of the present disclosure. In the case of disclosing a combination, each sub-combination of the elements of the combination is also specifically disclosed, and the sub-combinations are within the scope of the present disclosure. Conversely, in the case of separately disclosing different elements or groups of elements, their combinations are also disclosed. In the case where any element of the present disclosure is disclosed as having multiple alternatives, examples of the disclosure are also provided herein where each alternative is excluded individually or in any combination with other alternatives; more than one element of the present disclosure can have such exclusions, and all combinations of elements having such exclusions are thus disclosed.

[0202] Nucleotides can be represented by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written left to right in a 5' to 3' orientation. Nucleotides are referred to herein by their generally known single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0203] Amino acid sequences are written left to right in an amino to carboxyl orientation. Amino acids can be represented herein by their generally known three-letter symbols or single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature.

[0204] The term "about" is used herein to mean approximate, roughly, around, or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the recited numerical values. Generally, the term "about" can modify a numerical value by varying it up or down (raising or lowering) by, for example, 10%.

[0205] As used herein, the term "about" applied to one or more target values refers to a value similar to the stated reference value. In certain aspects, unless otherwise stated or apparent from the context (unless the number would exceed 100% of a possible value), the term "about" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value in either direction (greater than or less than).

[0206] The terms "administration," "administering," and their grammatical variants refer to introducing a composition, such as an EV (e.g., exosome) of the present disclosure, into a subject by a pharmaceutically acceptable route. Introducing a composition, such as an EV (e.g., exosome) of the present disclosure, into a subject is by any suitable route, including intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, periocular, or topical. Administration includes self-administration and administration by another person. A suitable route of administration enables the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into a subject's vein.

[0207] As used herein, the term "agonist" refers to a molecule that binds to a receptor and activates the receptor to produce a biological response. The receptor can be activated by endogenous or exogenous agonists. Non-limiting examples of endogenous agonists include hormones, neurotransmitters, and cyclic dinucleotides. Non-limiting examples of exogenous agonists include drugs, small molecules, and cyclic dinucleotides. An agonist can be a full, partial, or inverse agonist.

[0208] As used herein, the term "antagonist" refers to a molecule that, upon binding to a receptor, blocks or attenuates an agonist-mediated response and does not itself initiate a biological response. Many antagonists achieve their efficacy by competing with an endogenous ligand or substrate at a structurally defined binding site on the receptor. Non-limiting examples of antagonists include alpha blockers, beta blockers, and calcium channel blockers. Antagonists can be competitive, non-competitive, or uncompetitive antagonists.

[0209] As used herein, the term "extracellular vesicle" or "EV" refers to vesicles of cellular origin that contain a membrane surrounding an internal space (lumen). Extracellular vesicles include all membrane-bound vesicles that are smaller in diameter than the cell from which they are derived. Generally, extracellular vesicles have a diameter ranging from 20 nm to 1000 nm and can contain various payloads within the internal space (i.e., lumen), displayed on the outer or luminal surface of the extracellular vesicle, and / or transmembrane. Payloads can include, for example, nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and not limitation, extracellular vesicles include apoptotic bodies, cell debris, vesicles derived from cells by direct or indirect manipulation (e.g., by sequential extrusion or treatment with alkaline solutions), vesicularized organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, and / or cultured cells. In some aspects, the EVs contain the scaffold proteins disclosed herein.

[0210] As used herein, the term "exosome" refers to small (20 nm to 300 nm in diameter, more preferably 40 nm - 200 nm in diameter) extracellular vesicles (EVs) of cellular origin that contain a membrane surrounding an internal space (lumen) and, in some aspects, are produced by cells (e.g., producer cells) such as by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes are a type of extracellular vesicle (EV). In some aspects, exosomes contain lipids or fatty acids and polypeptides and optionally contain payloads (e.g., bioactive molecules such as therapeutic agents), acceptors (e.g., targeting moieties), polynucleotides (e.g., nucleic acids, RNA or DNA), sugars (e.g., monosaccharides, polysaccharides or glycans), or other molecules. Exosomes can be derived from producer cells and isolated from producer cells based on their size, density, biochemical parameters, or combinations thereof. In some aspects, exosomes contain the scaffold proteins of the present disclosure. In some aspects, the exosomes of the present disclosure are produced by cells expressing one or more transgenic products.

[0211] As used herein, the term "exosomal lumen protein" refers to a scaffolding protein, i.e., a protein such as MARCKS, MARKSL1, BASP1, or any functional fragment thereof, any variant thereof, any derivative thereof, or any combination thereof that attaches or associates with the luminal surface of an EV (e.g., exosome), and that is suitable for use as a scaffold to target a payload (e.g., a bioactive molecule (e.g., a therapeutic protein)) to the luminal surface of an EV (e.g., exosome).

[0212] As used herein, the term "nanovesicle" refers to small (having a diameter between 20 nm and 250 nm, e.g., a diameter of 30 nm - 150 nm) vesicles of cellular origin that contain a membrane surrounding an internal space and are produced from a cell (e.g., a producer cell) by direct or indirect manipulation such that the producer cell without such manipulation does not produce nanovesicles. Suitable manipulations for causing a producer cell to produce nanovesicles include, but are not limited to, sequential extrusion, treatment with an alkaline solution, sonication, or combinations thereof. In some cases, the production of nanovesicles can result in the disruption of the producer cell. In some aspects, the population of nanovesicles described herein is substantially free of vesicles derived from the producer cell by budding directly from the plasma membrane or by fusion of late endosomes with the plasma membrane. In some aspects, nanovesicles contain lipids or fatty acids and polypeptides and optionally contain a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecules. Once nanovesicles are derived from a producer cell according to the manipulation, the nanovesicles can be isolated from the producer cell based on their size, density, biochemical parameters, or combinations thereof. In some aspects, the nanovesicles can contain the scaffolding proteins disclosed herein.

[0213] As used herein, the term "lumen-engineered EV" refers to an EV (e.g., exosome) in which the luminal surface or the composition of the lumen of the membrane of the EV (e.g., exosome) is modified such that the luminal surface or the lumen of the engineered EV (e.g., exosome) is different from the luminal surface or the lumen of the EV (e.g., exosome) prior to modification or of a naturally occurring EV (e.g., exosome).

[0214] Engineering can be directly in the lumen (i.e., the void within the EV) or in the membrane of the EV (e.g., exosome), especially on the luminal surface of the EV, so that the lumen and / or luminal surface of the EV (e.g., exosome) are altered. For example, the composition of proteins, lipids, small molecules, carbohydrates, etc. in the membrane is modified, so that the luminal surface of the EV (e.g., exosome) is modified. Similarly, the content of the lumen can be modified. The composition can be altered by chemical, physical or biological methods, or by being produced by cells previously modified by chemical, physical or biological methods. Specifically, the composition can be altered by genetic engineering or by being produced by cells previously modified by genetic engineering. In some aspects, the lumen-engineered EVs (e.g., lumen-engineered exosomes) contain exogenous proteins (i.e., proteins that are not naturally expressed by the EVs (e.g., exosomes)) or fragments or variants thereof, and the exogenous proteins or fragments or variants thereof can be exposed to the luminal surface or lumen of the EV (e.g., exosome), or can be the anchor points (attachments) of the parts exposed on the inner layer of the EV (e.g., exosome). In other aspects, the lumen-engineered EVs (e.g., lumen-engineered exosomes) contain a higher expression of natural EV (e.g., exosome) proteins (e.g., scaffold proteins) or fragments or variants thereof, and the proteins or fragments or variants thereof can be exposed to the lumen of the EV (e.g., exosome), or can be the anchor points (attachments) of the parts exposed on the luminal surface of the EV (e.g., exosome).

[0215] As used herein, the term "surface-engineered EV" refers to an EV whose outer surface is modified in its composition such that the outer surface of the engineered EV is different from the outer surface of the EV before modification or the naturally occurring EV.

[0216] As used herein, the term "surface-engineered exosome" refers to an exosome whose outer surface is modified in its composition such that the outer surface of the engineered exosome is different from the outer surface of the exosome before modification or the naturally occurring exosome.

[0217] As used herein, the term "modified", when used in the context of EVs (e.g., exosomes) described herein, refers to altering and / or engineering the EV (e.g., exosome) and / or its producer cell such that the modified EV (e.g., exosome) is different from a naturally occurring EV (e.g., exosome). In some aspects, the modified EVs (e.g., exosomes) described herein comprise a membrane that is different in composition in terms of proteins, lipids, small molecules, carbohydrates, etc. compared to the membrane of a naturally occurring EV (e.g., exosome). For example, the membrane comprises a higher density or amount of proteins of a naturally occurring EV (e.g., exosome) and / or the membrane comprises proteins that do not naturally occur in EVs (e.g., exosomes). In certain aspects, such modifications to the membrane alter the outer surface of the EV (e.g., exosome (e.g., surface-engineered EVs and exosomes described herein)). In certain aspects, such modifications to the membrane alter the luminal surface of the EV (e.g., exosome (e.g., lumen-engineered EVs and exosomes described herein)).

[0218] For example, the composition of the proteins, lipids, small molecules, carbohydrates, etc. in the lumen is modified. The composition can be altered by chemical, physical, or biological methods, or by being produced by a cell that has been previously modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering or by being produced by a cell that has been previously modified by genetic engineering.

[0219] As used herein, the term "modified" of a protein, e.g., as in "modified protein" or "protein modification" or their grammatical variants, refers to a protein that has at least 15% identity with the non-mutated amino acid sequence of the protein. Modifications of a protein include fragments or variants of the protein. Modifications of a protein also include chemical or physical modifications of fragments or variants of the protein. In some aspects, the modified protein retains at least one physiological function of the unmodified protein.

[0220] As used herein, the term "fragment" of a protein (e.g., a bioactive molecule disclosed herein such as a therapeutic protein or a scaffold protein) refers to a protein that is shorter than the naturally occurring sequence, e.g., a protein with an N-terminal and / or C-terminal deletion compared to the naturally occurring protein.

[0221] As used herein, the term "functional fragment" refers to a protein fragment that retains the function of the protein. Thus, in some aspects, functional fragments of scaffold proteins such as MARCKS, MARCKSL1, or BASP1 disclosed herein retain the ability to anchor bioactive molecules on the luminal surface of EVs (e.g., exosomes).

[0222] In this sense, whether a fragment is a functional fragment can be evaluated by any method known in the art for determining the protein content of EVs (e.g., exosomes), including Western blotting, FACS analysis, and fusion of the fragment with a self-fluorescent protein (such as GFP). In certain aspects, a fragment of MARCKS, MARCKSL1, or BASP1 retains, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the ability of the naturally occurring MARCKS, MARCKSL1, or BASP1 to anchor a payload (e.g., a bioactive molecule) to the inner lumen surface or outer surface of an EV (e.g., an exosome). In certain aspects, the ability of a variant of MARCKS, MARCKSL1, BASP1, or a fragment of MARCKS, MARCKSL1, or BASP1 is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the ability of MARCKS, MARCKSL1, or BASP1 to anchor a payload (e.g., a bioactive molecule) to the inner lumen surface or outer surface of an EV (e.g., an exosome). This ability can be evaluated, for example, by a fluorescently labeled variant in the assays described in the experimental section.

[0223] As used herein, the term "derivative" refers to an EV (e.g., an exosome), a component (e.g., a protein or lipid), a scaffold protein, or a payload of the present disclosure, e.g., a bioactive molecule (such as a polypeptide, polynucleotide, lipid, carbohydrate, antibody, or fragment thereof) that has been chemically or enzymatically modified.

[0224] As used herein, the term "variant" of a protein refers to a protein that shares a certain structure (e.g., amino acid sequence identity) and functional identity with another protein when compared by methods known in the art (e.g., by sequence alignment). For example, variants of a protein can include substitutions, insertions, deletions, frameshifts, or rearrangements in another protein. In some aspects, a variant of a protein retains at least one physiological function of the non-variant protein.

[0225] In a particular aspect, the variant is a variant protein that has at least 70% identity with the full-length, mature MARCKS, MARCKSL1, BASP1, or a fragment of MARCKS, MARCKSL1, or BASP1.

[0226] In some aspects, variants of MARCKS or variants of fragments thereof share at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence identity with MARCKS according to SEQ ID NO:1 or a functional fragment thereof, as determined, for example, by pairwise alignment using the Needleman-Wunsch algorithm.

[0227] In some aspects, variants of MARCKSL1 or variants of fragments thereof share at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence identity with MARCKSL1 according to SEQ ID NO:2 or a functional fragment thereof, as determined, for example, by pairwise alignment using the Needleman-Wunsch algorithm.

[0228] In some aspects, variants of BASP1 or variants of fragments thereof share at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence identity with BASP1 according to SEQ ID NO:3 or a functional fragment thereof, as determined, for example, by pairwise alignment using the Needleman-Wunsch algorithm.

[0229] In some aspects, variants of a scaffolding protein (e.g., MARCKS, MARCKSL1 or BASP1), functional fragments of a scaffolding protein, or variants of functional fragments of a scaffolding protein are derivatives.

[0230] In each of the above cases, variants of a scaffolding protein or variants of fragments thereof (e.g., MARCKS, MARCKSL1, BASP1 or variants of fragments of MARCKS, MARCKSL1 or BASP1) retain the ability to anchor a payload (e.g., a bioactive molecule) to the luminal or outer surface of an EV (e.g., an exosome).

[0231] The recitation of any protein provided herein encompasses functional variants of the protein. The term "functional variant" of a protein refers to a variant of a protein that retains the ability to anchor a bioactive molecule to the luminal or outer surface of an EV (e.g., an exosome). In certain aspects, the ability of a functional variant of MARCKS, MARCKSL1, BASP1 or a fragment of MARCKS, MARCKSL1 or BASP1 is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% of the ability of MARCKS, MARCKSL1 or BASP1 to anchor a payload (e.g., a bioactive molecule) to the luminal or outer surface of an EV (e.g., an exosome).

[0232] Naturally occurring variants are referred to as "allelic variants" and are one of several alternative forms of a gene that occupy a given locus on the chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at the polynucleotide and / or polypeptide level and are included in the present disclosure. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.

[0233] Using known methods of protein engineering and recombinant DNA technology, variants can be produced to improve or alter the properties of a polypeptide. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein with little or no loss of biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993) (incorporated herein by reference in its entirety) reported a variant KGF protein that still had heparin-binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, after deletion of 8-10 amino acid residues from the carboxyl terminus of interferon γ, the protein exhibited up to ten-fold higher activity. (Dobeli et al., J. Biotechnology 7:199-216 (1988), incorporated herein by reference in its entirety).

[0234] In addition, a large body of evidence indicates that variants generally retain biological activity similar to that of the naturally occurring protein. For example, Gayle and colleagues (J. Biol. Chem 268:22105-22111 (1993), incorporated herein by reference in its entirety) performed an extensive mutagenesis analysis of the human cytokine IL-1α. They generated over 3,500 individual IL-1α mutants using random mutagenesis, with an average of 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at each possible amino acid position. The researchers found that "most of the molecule can be altered with little or no effect on [binding or biological activity]." (See abstract). In fact, out of the over 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins with significantly different activity from the wild type.

[0235] As described above, variants or derivatives include, for example, modified polypeptides. In some aspects, variants or derivatives of, for example, polypeptides, polynucleotides, lipids, glycoproteins are the result of chemical modification and / or endogenous modification. In some aspects, variants or derivatives are the result of in vivo modification. In some aspects, variants or derivatives are the result of in vitro modification. In other aspects, variants or derivatives are the result of intracellular modification in the producer cell.

[0236] Modifications present in variants and derivatives include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of a covalent cross-link, formation of cysteine, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, polyethylene glycolylation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of an amino acid to a protein mediated by transfer RNA such as arginylation and ubiquitination.

[0237] The term "amino acid substitution" refers to the replacement of an amino acid residue present in a parental or reference sequence (e.g., a wild-type sequence) with another amino acid residue. The amino acids in a parental or reference sequence (e.g., a wild-type polypeptide sequence) can be replaced, for example, by chemical peptide synthesis or by recombinant methods known in the art. Thus, reference to a "substitution at position X" means that the amino acid present at position X is replaced with an alternative amino acid residue. In some aspects, the substitution pattern can be described according to the schema AnY, where A is the single-letter code corresponding to the amino acid that is naturally or initially present at position n, and Y is the substituted amino acid residue. In other aspects, the substitution pattern can be described according to the schema An(YZ), where A is the single-letter code corresponding to the amino acid residue that substitutes the amino acid that is naturally or initially present at position n, and Y and Z are alternative substituted amino acid residues that can replace A.

[0238] As used herein, the term "antibody" encompasses immunoglobulins and their fragments, whether produced naturally or in part or in whole synthetically. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" also includes polypeptides that contain framework regions from immunoglobulin genes or fragments thereof that specifically bind and recognize an antigen. The use of the term antibody is meant to include intact antibodies, polyclonal, monoclonal, and recombinant antibodies, fragments thereof, and also includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as, for example, scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. Antibodies include bispecific and multispecific antibodies, provided that they exhibit the desired biological activity or function. In some aspects of the present disclosure, the payload is a bioactive molecule that includes an antibody or an antigen-binding fragment thereof. In some aspects, the antibody (e.g., the bioactive molecule of the present disclosure) is a nanobody.

[0239] The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to, for example, an antibody covalently linked to a therapeutic agent (sometimes referred to herein as an agent, drug, or active pharmaceutical ingredient). In some aspects of the present disclosure, the payload is a bioactive molecule that includes an antibody-drug conjugate.

[0240] "Conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a substitution is considered conservative if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family. In another aspect, an amino acid string can be conservatively substituted with a structurally similar string having a different order and / or composition of side chain family members.

[0241] As used herein, the term "conservative" refers, respectively, to nucleotide or amino acid residues of a polynucleotide sequence or polypeptide sequence that are those nucleotide or amino acid residues that are present unchanged at the same position in two or more sequences being compared. A relatively conservative nucleotide or amino acid is a nucleotide or amino acid that is conserved in more related sequences compared to nucleotides or amino acids that occur elsewhere in the sequence.

[0242] In some aspects, two or more sequences are said to be "fully conservative" or "identical" if they have 100% identity to each other. In some aspects, two or more sequences are considered to be "highly conservative" if they have at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to each other.

[0243] In some aspects, two or more sequences are considered to be "highly conservative" if they have about 70% identity, about 80% identity, about 90% identity, or about 95%, about 98%, or about 99% identity to each other. In some aspects, two or more sequences are considered to be "conservative" if they have at least 30% identity, at least 40% identity, at least 50% identity, at least 60% identity, at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to each other. In some aspects, two or more sequences are considered to be "conservative" if they have about 30% identity, about 40% identity, about 50% identity, up to about 60% identity, about 70% identity, about 80% identity, about 90% identity, about 95% identity, at least 98% identity, or at least 99% identity to each other. The conservativeness of a sequence can apply to the full length of a polynucleotide or polypeptide, or can apply to a portion, region, or feature thereof.

[0244] As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, such as between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" indicates an evolutionary relationship between two molecules. Thus, two homologous molecules will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses identity and similarity.

[0245] In some aspects, polymeric molecules are considered to be "homologous" to each other if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the monomers are identical (identical monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0246] In the context of the present disclosure, substitutions are made at the nucleic acid level (even when they are referred to as amino acid substitutions), i.e., substituting an amino acid residue with an alternative amino acid residue is effected by substituting the codon encoding the first amino acid with the codon encoding the second amino acid.

[0247] As used herein, the term "identity" refers to overall monomer conservation between polymer molecules, e.g., between polypeptide molecules or polynucleotide molecules (such as DNA molecules and / or RNA molecules). The term "identical", without any other qualifier, e.g., protein A is identical to protein B, means that the sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, "70% identical" is equivalent to describing them as having, for example, "70% sequence identity".

[0248] For example, the percent identity of two polypeptide sequences can be calculated by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second polypeptide sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In some aspects, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. Then the amino acids at the corresponding amino acid positions are compared.

[0249] When the position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to obtain an optimal alignment of the two sequences and the length of each gap. Mathematical algorithms can be used to effect sequence comparison and to determine the percent identity between two sequences.

[0250] Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are described in the following references: Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24:307-31 (1988); Higgins and Sharp, Gene 73:15 237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992); and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) [Altschul 20 et al., J. Mol. Biol. 215:403-10 (1990)] is available from multiple sources including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) of the United States and the Internet, and is used in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. BLAST and instructions on how to use the program to determine sequence identity can be accessed at the official website of the NCBI (National Center for Biotechnology Information) of the NIH (National Institutes of Health) of the United States.

[0251] Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa. Sequence alignment can be performed using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, etc.

[0252] The different regions within a single polynucleotide or polypeptide target sequence aligned with a polynucleotide or polypeptide reference sequence can each have their own percentage of sequence identity. It should be noted that the percentage of sequence identity values are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that the length values will always be integers.

[0253] In certain embodiments, the percent identity (ID%) of a first amino acid sequence (or nucleotide sequence) to a second amino acid (or nucleotide sequence) is calculated as ID% = 100 x (Y / Z), where Y is the number of amino acid residues (or nucleobases) scored as identical matches in an alignment of the first and second sequences (such as by visual inspection or using a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0254] Those skilled in the art will understand that the sequence alignments generated for calculating percent sequence identity are not limited to binary sequence-sequence comparisons driven solely by primary sequence data. It should also be understood that sequence alignments can be generated by integrating sequence data with data from heterologous sources such as structural data (e.g., crystal protein structures), functional data (e.g., positions of mutations), or phylogenetic data. A suitable program for integrating heterologous data to generate multiple sequence alignments is T-Coffee, which is available from www.tcoffee.org and / or, for example, from the EBI. It should also be understood that the final alignment used for calculating percent sequence identity can be performed automatically or manually (curated).

[0255] As used herein, the term "similarity" refers to the overall relatedness between polymeric molecules, such as between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. The percent similarity between polymeric molecules can be calculated in the same manner as the percent identity, except that the calculation of percent similarity takes into account conservative substitutions known in the art. It should be understood that the percent similarity depends on the comparison criteria used, i.e., whether amino acids are compared, for example, based on evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or a combination thereof.

[0256] As used herein, the term "producer cell" refers to a cell used to produce EVs (e.g., exosomes). Producer cells can be cells cultured in vitro or in vivo. Producer cells include, but are not limited to, cells known to be effective in producing EVs (e.g., exosomes), such as, for example, HEK293 cells, Chinese hamster ovary (CHO) cells, and mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, neuronal progenitor cells, Amniotic fluid cells, adipose mesenchymal stem cells, RPTEC / TERT1 cells. In some aspects, the producer cells are not antigen-presenting cells. In some aspects, the producer cells are not dendritic cells, B cells, mast cells, macrophages, neutrophils, Kupffer-Browicz cells, cells derived from any of these cells, or any combination thereof.

[0257] As used herein, the terms "isolate", "isolated", and "isolating" or "purify", "purified", and "purifying" and "extracted" and "extracting" and their grammatical variants are used interchangeably and refer to a preparation state (e.g., various known or unknown amounts and / or concentrations) of the desired EVs (e.g., exosomes) that have undergone one or more purification processes (e.g., selection or enrichment of the desired EV (e.g., exosome) preparation). In some embodiments, as used herein, isolation or purification is the process of removing, partially removing (e.g., a portion of) EVs (e.g., exosomes) from a sample containing producer cells. In some embodiments, the isolated EV (e.g., exosome) composition has no detectable unwanted activity, or the level or amount of unwanted activity is equal to or lower than an acceptable level or amount.

[0258] In other embodiments, the isolated EV (e.g., exosome) composition has an amount and / or concentration of the desired EVs (e.g., exosomes) that is equal to or higher than an acceptable amount and / or concentration. In other aspects, the isolated EV (e.g., exosome) composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition is obtained. This enrichment can be at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than about 99.9999% compared to the starting material.

[0259] In some aspects, the isolated EV (e.g., exosome) preparation is substantially free of residual biological products (e.g., contaminants). In some aspects, the isolated EV (e.g., exosome) preparation is about 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological substances. Residual biological products can include non-biological materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. In certain aspects, the isolated EV (e.g., exosome) preparation is about 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any macromolecules, e.g., free of any nucleic acids, proteins, lipids, and / or carbohydrates. Substantially free of residual biological products can also mean that the EV (e.g., exosome) composition does not contain detectable producer cells and only the EV (e.g., exosome) is detectable.

[0260] The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound. The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", and their grammatical variants encompass any agent approved by a regulatory agency of the Federal Government of the United States or listed in the United States Pharmacopeia for use in animals (including humans), as well as any vehicle or diluent that does not cause significant irritation to the subject and does not abrogate the biological activity and properties of the administered compound. Excipients and carriers that are useful in the preparation of pharmaceutical compositions and are generally safe, non-toxic, and desired are included.

[0261] As used herein, the term "payload" refers to any molecule that can be attached to a scaffold of the present disclosure and subsequently anchored to the membrane of an EV (e.g., exosome) of the present disclosure. In some embodiments, the payload is attached to the luminal surface of the EV (e.g., exosome) membrane. The term payload encompasses bioactive molecules, such as molecules that may have a therapeutic and / or prophylactic effect, as well as diagnostic molecules. Thus, the term payload also encompasses detectable moieties, such as radionuclides, fluorescent molecules, contrast agents, tags, or molecular entities that can be recognized by a detectable moiety (e.g., a ligand or a tag).

[0262] As used herein, the term payload is equivalent to the term "cargo" and can be used interchangeably with the term "cargo". Thus, "cargo protein" or "cargo peptide" refers to a specific type of payload molecule (protein and peptide, respectively) attached to a scaffold of the present disclosure.

[0263] As used herein, the term "bioactive molecule" refers to any molecule that can be attached to an EV (e.g., exosome) via the scaffolds of the present disclosure, wherein the molecule can have a therapeutic or prophylactic effect in a subject in need thereof, or affect the homeostasis of cells or tissues in a subject. Non-limiting examples of bioactive molecules that can be introduced into EVs (e.g., exosomes) and / or producer cells include, for example, therapeutic agents such as nucleotides (e.g., nucleotides containing a detectable moiety or a toxin or disrupting transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes or RNA molecules with regulatory functions such as miRNAs, dsDNAs, lncRNAs, and siRNAs), amino acids (e.g., amino acids containing a detectable moiety or a toxin or disrupting translation), polypeptides (e.g., enzymes or antibodies), lipids, carbohydrates, viruses and virus particles (e.g., adeno-associated viruses and virus particles, retroviruses, adenoviruses, etc.), and small molecules (e.g., small molecule drugs and toxins), including small molecule STING agonists, including cyclic dinucleotides such as ML-RR S2 and 3'-3’cAIMPdFSH). In certain aspects, the payload includes an antigen. In certain aspects, when fused to a scaffold protein disclosed herein, a bioactive molecule, such as a therapeutic agent (e.g., a human therapeutic agent), retains its biological function, such as its therapeutic function. In some aspects, a bioactive molecule can have at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, or at least 500 amino acids. In some aspects, a bioactive molecule can have at least 5 amino acids. In some aspects, a bioactive molecule can have at least 10 amino acids. In some aspects, a bioactive molecule can have at least 50 amino acids. In some aspects, a bioactive molecule can have at least 100 amino acids. In some aspects, a bioactive molecule can have at least 5 amino acids, such as 5 - 500 amino acids, 5 - 450 amino acids, 5 - 400 amino acids, 5 - 350 amino acids, 5 - 300 amino acids, 5 - 250 amino acids, 5 - 200 amino acids, 5 - 150 amino acids, 5 - 100 amino acids, 5 - 90 amino acids, 5 - 80 amino acids, 5 - 70 amino acids, 5 - 60 amino acids, 5 - 50 amino acids, 5 - 40 amino acids, 5 - 30 amino acids, 5 - 20 amino acids, 5 - 15 amino acids, or 5 - 10 amino acids.In some aspects, the bioactive molecule can have at least 10 amino acids, such as 10 - 500, 10 - 450, 10 - 400, 10 - 350, 10 - 300, 10 - 250, 10 - 200, 10 - 150, 10 - 100, 10 - 90, 10 - 80, 10 - 70, 10 - 60, 10 - 50, 10 - 40, 10 - 30, 10 - 20, or 10 - 15 amino acids. In some aspects, the bioactive molecule can have at least 50 amino acids, such as 50 - 500, 50 - 450, 50 - 400, 50 - 350, 50 - 300, 50 - 250, 50 - 200, 50 - 150, 50 - 100, 50 - 90, 50 - 80, 50 - 50, or 50 - 60 amino acids. In some aspects, the bioactive molecule can have at least 100 amino acids, such as 100 - 500, 100 - 450, 100 - 400, 100 - 350, 100 - 300, 100 - 250, 100 - 200, or 100 - 150 amino acids. In other aspects, the bioactive molecule has at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 300, at least 400, or at least 500 nucleotides. In other aspects, the bioactive molecule has at least two nucleotides, such as 2 - 1000, 2 - 900, 2 - 500, 2 - 300, or 2 - 50 nucleotides. In other aspects, the bioactive molecule has at least 10 nucleotides, such as 10 - 1000, 10 - 900, 10 - 500, 10 - 300, or 10 - 50 nucleotides. In other aspects, the bioactive molecule has at least 15 nucleotides, such as 15 - 1000, 15 - 900, 15 - 500, 15 - 300, or 15 - 50 nucleotides. In other aspects, the bioactive molecule has at least 20 nucleotides, such as 20 - 1000, 20 - 900, 20 - 500, 20 - 300, or 20 - 50 nucleotides. In other aspects, the bioactive molecule has at least 50 nucleotides, such as 50 - 1000, 50 - 900, 50 - 500, 50 - 300, or 50 - 100 nucleotides.In other aspects, the bioactive molecule has at least 100 nucleotides, such as 100 - 10000, 100 - 9000, 100 - 5005, 100 - 3000, or 100 - 500 nucleotides. In other aspects, the bioactive molecule has at least 200 nucleotides, such as 200 - 10000, 200 - 9000, 200 - 5000, 200 - 3000, or 200 - 500 nucleotides. In other aspects, the bioactive molecule has at least 500 nucleotides, such as 500 - 10000, 500 - 9000, 500 - 5000, or 500 - 3000 nucleotides. As used herein, the term "antigen" refers to any agent that, when introduced into a subject, elicits an immune response (cellular or humoral) against itself. In some aspects, the payload molecule is covalently linked to an EV (e.g., exosome). In other aspects, the payload includes an adjuvant.

[0264] A "recombinant" polypeptide or protein refers to a polypeptide or protein produced by recombinant DNA technology. For the purposes of this disclosure, recombinantly produced polypeptides and proteins expressed in engineered host cells and isolated are considered to be natural or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any suitable technique. The polypeptides disclosed herein can be recombinantly produced using methods known in the art. Alternatively, the proteins and peptides disclosed herein can be chemically synthesized. In some aspects of the present disclosure, the scaffold protein present in an EV (e.g., exosome) is recombinantly produced by overexpressing the scaffold protein in a producer cell, and the overexpression of the scaffold protein results in a significant increase in the level of the scaffold protein in the resulting EV (e.g., exosome) relative to the level of the scaffold protein present in the EV (e.g., exosome) of a producer cell that does not overexpress such a scaffold protein.

[0265] As used herein, the term "anchor" or "anchoring" a bioactive molecule to the luminal or outer surface of an EV (e.g., exosome) of the present disclosure via a scaffold protein refers to covalently attaching the bioactive molecule to a portion of a scaffold molecule located on the luminal or outer surface of the EV (e.g., exosome), respectively.

[0266] The terms "associated", "associating" and their grammatical variants are used interchangeably and refer to a first moiety (e.g., a first amino acid sequence or nucleotide sequence) being covalently or non-covalently linked to a second moiety (e.g., a second amino acid sequence or nucleotide sequence), respectively. The first moiety can be directly linked or juxtaposed to the second moiety, or, optionally, intervening moieties can covalently link the first moiety to the second moiety. In some embodiments, the term "associated" includes myristoylation, ionic interactions, or any combination thereof.

[0267] As used herein, the terms "linked" or "fused" refer to the fusion of a first moiety to a second moiety at the C-terminus or N-terminus via a peptide bond or via a linker of one or more amino acids. The terms "linked" or "fused" also include inserting the entire first moiety (or second moiety) between any two points (e.g., amino acids) within, for example, the second moiety (or first moiety). In one aspect, the first moiety is linked to the second moiety via a peptide bond or a linker. The first moiety can be linked to the second moiety via a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (for polypeptide chains) or a nucleotide or nucleotide chain (for nucleotide chains) or any chemical moiety (for polypeptide or polynucleotide chains or any chemical molecule). The term "linked" can also be denoted by a hyphen (-). In some aspects, a scaffold protein on an EV (e.g., an exosome) can be linked or fused to a payload (e.g., a bioactive molecule).

[0268] As used herein, "mammalian subject" includes all mammals, including but not limited to humans, domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0269] The terms "individual", "subject", "host", and "patient" and their variants are used interchangeably herein and refer to any mammalian subject, particularly a human, to whom diagnosis, treatment, or therapy is desired. The methods described herein are applicable to human therapy and veterinary applications. In some aspects, the subject is a mammal, and in other aspects, the subject is a human.

[0270] As used herein, the term "substantially free" means that, by mass / volume (m / v) percentage concentration, a sample containing EVs (e.g., exosomes) contains less than 10% macromolecules (e.g., contaminants). Some fractions may contain less than about 0.001%, less than about 0.01%, less than about 0.05%, less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, or less than about 10% (m / v) macromolecules.

[0271] As used herein, the term "macromolecule" means nucleic acid, foreign protein, lipid, carbohydrate, metabolite (e.g., polymeric metabolite), or a combination thereof.

[0272] As used herein, the term "conventional EV protein" refers to a protein previously known to be enriched in EVs.

[0273] As used herein, the term "conventional EV (e.g., exosome) protein" means a protein previously known to be enriched in exosomes, including but not limited to CD9, CD63, CD81, PDGFR, GPI-anchored proteins, lactadherin LAMP2 and LAMP2B, fragments thereof, or peptides that bind thereto. For the avoidance of doubt, PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporters, or fragments or variants thereof are not conventional EV (e.g., exosome) proteins.

[0274] As used herein, the term "pharmaceutical composition" refers to one or more of the compounds described herein, such as, for example, EVs such as the exosomes of the present disclosure, mixed or admixed or suspended with one or more other chemical components, such as pharmaceutically acceptable carriers and excipients. One purpose of the pharmaceutical composition is to facilitate the administration of a formulation of EVs (e.g., exosomes) to a subject.

[0275] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length (including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof). The term refers to the primary structure of a molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-stranded, double-stranded, and single-stranded ribonucleic acid ("RNA"). It also includes modified (e.g., by alkylation and / or by capping) and unmodified forms of polynucleotides. More particularly, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA (whether spliced or unspliced), any other type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholino polymers, as well as other synthetic sequence-specific nucleic acid polymers, provided that the polymers contain nucleobases present in a configuration that permits base pairing and base stacking, such as the bases present in DNA and RNA. In certain aspects, the polynucleotide includes mRNA. In other aspects, the mRNA is synthetic mRNA. In some aspects, the synthetic mRNA contains at least one unnatural nucleobase. In some aspects, all nucleobases of a certain class have been replaced by unnatural nucleobases (e.g., all uridines in the polynucleotides disclosed herein can be replaced by an unnatural nucleobase (e.g., 5-methoxyuridine)). In some aspects of the present disclosure, the bioactive molecule is a polynucleotide.

[0276] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may contain modified amino acids. These terms also encompass polymers of amino acids that have been naturally modified or modified by intervening operations; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification (such as conjugation to a labeling component). The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and sarcosine) and other modifications known in the art. In some aspects of the present disclosure, the payloads attached to EVs (e.g., exosomes), such as bioactive molecules, are polypeptides, such as antibodies or their derivatives, such as ADCs, PROTACs, toxins, fusion proteins, or enzymes.

[0277] As used herein, the term "polypeptide" refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments of the foregoing, and other equivalents, variants, and analogs. Polypeptides can be single polypeptides or multimolecular complexes, such as dimers, trimers, or tetramers. They can also contain single-chain or multi-chain polypeptides. Most commonly, disulfide linkages are present in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some aspects, the length of a "peptide" can be less than or equal to 50 amino acids, e.g., a length of about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some aspects, the length of a "peptide" can be at least about 2 to about 50, at least about 3 to about 50, at least about 4 to about 50, at least about 5 to about 50, at least about 10 to about 50, at least about 15 to about 50, at least about 20 to about 50, at least about 25 to about 50, at least about 30 to about 50, at least about 35 to about 50, at least about 40 to about 50, or at least about 45 to about 50 amino acids.

[0278] As used herein, the term "scaffold protein of the present disclosure" or grammatical variants thereof refers to

[0279] (i) a protein (naturally expressed, chemically or enzymatically synthesized, or recombinantly produced) located on the inner lumen surface of an EV (e.g., exosome), such as MARCKS, MARKSL1, or BASP1;

[0280] (ii) any functional fragment of (i);

[0281] (iii) any functional variant of (i)-(ii);

[0282] (iv) Any derivatives of (i)-(iii);

[0283] (v) Any peptide corresponding to a domain of the protein from (i) or a combination thereof, or a molecule comprising said peptide, which peptide can bind to the luminal surface of an EV (e.g., exosome);

[0284] (vi) Any peptide derived from a motif of the protein from (i), or a molecule comprising said peptide, which peptide can bind to the luminal surface of an EV (e.g., exosome);

[0285] (vii) Molecules of (i) to (vi) that comprise at least one unnatural amino acid;

[0286] (viii) Or any combination thereof,

[0287] which are suitable for use as scaffolds to target (attach) a payload (e.g., a bioactive molecule (e.g., a therapeutic protein)) to the luminal surface of an EV (e.g., exosome).

[0288] As used herein, the term "EVs (e.g., exosomes) of the present disclosure" or grammatical variants thereof refers to EVs (e.g., exosomes) that comprise at least one scaffold protein of the present disclosure.

[0289] As used herein, the term "producer cells of the present disclosure" or grammatical variants thereof refers to cells that can produce EVs (e.g., exosomes) of the present disclosure.

[0290] II. Extracellular vesicle proteins, e.g., exosome proteins

[0291] Some aspects of the present disclosure relate to the identification, use, and modification of EV (e.g., exosome) proteins (scaffold proteins) that are highly enriched in the luminal surface of EVs (e.g., exosomes). Such EV proteins or exosome proteins (scaffold proteins) can be identified by analyzing highly purified EVs (e.g., exosomes) using mass spectrometry or other methods known in the art.

[0292] The scaffold proteins of the present disclosure include various luminal proteins or membrane proteins that are enriched in the EV (e.g., exosome) membrane, such as transmembrane proteins (i.e., proteins that span the EV membrane through one or more transmembrane helices), integral proteins, and peripheral proteins (i.e., proteins that interact with the surface through electrostatic interactions and / or an anchoring moiety on the luminal surface). Specifically, the scaffold proteins of the present disclosure include, but are not limited to,

[0293] (1) Myristoylated alanine-rich C kinase substrate (MARCKS);

[0294] (2) Myristoylated alanine-rich C kinase substrate-like protein 1 (MARCKSL1); and

[0295] (3) Brain acid-soluble protein 1 (BASP1).

[0296] One or more EV (e.g., exosome) proteins (scaffold proteins) identified herein can be selectively used depending on the producer cell, production conditions, purification method, or intended application of the exosome.

[0297] EV (e.g., exosome) proteins enriched in the lumen (e.g., on the luminal surface of the EV membrane) of certain EV (e.g., exosomes) having a specific size range, targeting moiety, charge density, payload, etc., can be identified and used in certain aspects of the present disclosure.

[0298] In some aspects, more than one EV (e.g., exosome) protein disclosed herein (e.g., scaffold proteins such as MARCKS, MARKSL1, BASP1, any functional fragment, variant, or derivative thereof, or any combination thereof) can be used simultaneously, or can subsequently be used to generate or isolate the therapeutic EV (e.g., exosomes) of the present disclosure.

[0299] III. Lumen-engineered EV (e.g., exosomes)

[0300] Extracellular vesicles (EVs), e.g., exosomes, typically have a diameter of 20 nm to 1000 nm. Exosomes are small extracellular vesicles, typically having a diameter of 100 - 200 nm. EVs (e.g., exosomes) are composed of a limiting lipid bilayer and different sets of proteins and nucleic acids (Maas, S.L.N. et al., Trends. Cell Biol. 27(3):172 - 188 (2017)). EVs (e.g., exosomes) exhibit preferential uptake in discrete cell types and tissues, and their tropism can be directed by adding proteins that interact with receptors on the surface of target cells (Alvarez-Erviti, L., et al., Nat. Biotechnol. 29(4):341 - 345 (2011)).

[0301] Unlike antibodies, EVs (e.g., exosomes) can accommodate a large number of molecules attached to their surface, approximately thousands to tens of thousands of molecules per EV (e.g., exosome). Thus, conjugates or complexes containing EVs (e.g., exosomes) and payloads (e.g., bioactive molecules (e.g., therapeutic molecules)) represent a platform that delivers high concentrations of therapeutic or diagnostic compounds to discrete cell types while limiting the overall systemic exposure to the compound, which in turn reduces off-target toxicity. In addition, EVs (e.g., exosomes) offer the possibility of accommodating multiple payloads (e.g., bioactive molecules) in different compartments. For example, EVs (e.g., exosomes) can contain, for example, targeting moieties (the targeting moieties are attached to the outer surface of the EV and direct the EV to a certain target cell (e.g., cancer cell) or target tissue (e.g., liver or brain)), one or more therapeutic moieties (e.g., drugs) and / or one or more detectable moieties (e.g., contrast agents or radionuclides).

[0302] EVs (e.g., exosomes) can also contain different payloads, for example, therapeutic moieties (e.g., drugs) and / or detectable moieties (e.g., contrast agents or radionuclides) attached to the luminal surface of the EV. In addition, EVs (e.g., exosomes) can also contain one or more payloads in the lumen of the EV (e.g., exosome), for example, bioactive molecules (e.g., therapeutic agents, diagnostic reagents, adjuvants, etc.).

[0303] Thus, EVs (e.g., exosomes) provide a delivery format that combines multiple payloads (e.g., bioactive molecules) with the same or different functions at an extremely high density in a single delivery vehicle.

[0304] The EVs (e.g., exosomes) described herein are extracellular vesicles with a diameter of about 20 nm to 300 nm. In certain embodiments, the EVs (e.g., exosomes) of the present disclosure have a diameter of about 20 nm to 290 nm, 20 nm to 280 nm, 20 nm to 270 nm, 20 nm to 260 nm, 20 nm to 250 nm, 20 nm to 240 nm, 20 nm to 230 nm, 20 nm to 220 nm, 20 nm to 210 nm, 20 nm to 200 nm, 20 nm to 190 nm, 20 nm to 180 nm, 20 nm to 170 nm, 20 nm to 160 nm, 20 nm to 150 nm, 20 nm to 140 nm, 20 nm to 130 nm, 20 nm to 120 nm, 20 nm to 110 nm, 20 nm to 100 nm, 20 nm to 90 nm, 20 nm to 80 nm, 20 nm to 70 nm, 20 nm to 60 nm, 20 nm to 50 nm, 20 nm to 40 nm, 20 nm to 30 nm, 30 nm to 300 nm, 30 nm to 290 nm, 30 nm to 280 nm, 30 nm to 270 nm, 30 nm to 260 nm, 30 nm to 250 nm, 30 nm to 240 nm, 30 nm to 230 nm, 30 nm to 220 nm, 30 nm to 210 nm, 30 nm to 200 nm, 30 nm to 190 nm, 30 nm to 180 nm, 30 nm to 170 nm, 30 nm to 160 nm, 30 nm to 150 nm, 30 nm to 140 nm, 30 nm to 130 nm, 30 nm to 120 nm, 30 nm to 110 nm, 30 nm to 100 nm, 30 nm to 90 nm, 30 nm to 80 nm, 30 nm to 70 nm, 30 nm to 60 nm, 30 nm to 50 nm, 30 nm to 40 nm, 40 nm to 300 nm, 40 nm to 290 nm, 40 nm to 280 nm, 40 nm to 270 nm, 40 nm to 260 nm, 40 nm to 250 nm, 40 nm to 240 nm, 40 nm to 230 nm, 40 nm to 220 nm, 40 nm to 210 nm, 40 nm to 200 nm, 40 nm to 190 nm, 40 nm to 180 nm, 40 nm to 170 nm, 40 nm to 160 nm, 40 nm to 150 nm, 40 nm to 140 nm, 40 nm to 130 nm, 40 nm to 120 nm, 40 nm to 110 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 40 nm to 60 nm, 40 nm to 50 nm, 50 nm to 300 nm, 50 nm to 290 nm, 50 nm to 280 nm, 50 nm to 270 nm, 50 nm to 260 nm, 50 nm to 250 nm,50 nm to 240 nm, 50 nm to 230 nm, 50 nm to 220 nm, 50 nm to 210 nm, 50 nm to 200 nm, 50 nm to 190 nm, 50 nm to 180 nm, 50 nm to 170 nm, 50 nm to 160 nm, 50 nm to 150 nm, 50 nm to 140 nm, 50 nm to 130 nm, 50 nm to 120 nm, 50 nm to 110 nm, 50 nm to 100 nm, 50 nm to 90 nm, 50 nm to 80 nm, 50 nm to 70 nm, 50 nm to 60 nm, 60 nm to 300 nm, 60 nm to 290 nm, 60 nm to 280 nm, 60 nm to 270 nm, 60 nm to 260 nm, 60 nm to 250 nm, 60 nm to 240 nm, 60 nm to 230 nm, 60 nm to 220 nm, 60 nm to 210 nm, 60 nm to 200 nm, 60 nm to 190 nm, 60 nm to 180 nm, 60 nm to 170 nm, 60 nm to 160 nm, 60 nm to 150 nm, 60 nm to 140 nm, 60 nm to 130 nm, 60 nm to 120 nm, 60 nm to 110 nm, 60 nm to 100 nm, 60 nm to 90 nm, 60 nm to 80 nm, 60 nm to 70 nm, 70 nm to 300 nm, 70 nm to 290 nm, 70 nm to 280 nm, 70 nm to 270 nm, 70 nm to 260 nm, 70 nm to 250 nm, 70 nm to 240 nm, 70 nm to 230 nm, 70 nm to 220 nm, 70 nm to 210 nm, 70 nm to 200 nm, 70 nm to 190 nm, 70 nm to 180 nm, 70 nm to 170 nm, 70 nm to 160 nm, 70 nm to 150 nm, 70 nm to 140 nm, 70 nm to 130 nm, 70 nm to 120 nm, 70 nm to 110 nm, 70 nm to 100 nm, 70 nm to 90 nm, 70 nm to 80 nm, 80 nm to 300 nm, 80 nm to 290 nm, 80 nm to 280 nm, 80 nm to 270 nm, 80 nm to 260 nm, 80 nm to 250 nm, 80 nm to 240 nm, 80 nm to 230 nm, 80 nm to 220 nm, 80 nm to 210 nm, 80 nm to 200 nm, 80 nm to 190 nm, 80 nm to 180 nm, 80 nm to 170 nm, 80 nm to 160 nm, 80 nm to 150 nm, 80 nm to 140 nm, 80 nm to 130 nm, 80 nm to 120 nm, 80 nm to 110 nm, 80 nm to 100 nm, 80 nm to 90 nm, 90 nm to 300 nm, 90 nm to 290 nm, 90 nm to 280 nm,90 nm to 270 nm, 90 nm to 260 nm, 90 nm to 250 nm, 90 nm to 240 nm, 90 nm to 230 nm, 90 nm to 220 nm, 90 nm to 210 nm, 90 nm to 200 nm, 90 nm to 190 nm, 90 nm to 180 nm, 90 nm to 170 nm, 90 nm to 160 nm, 90 nm to 150 nm, 90 nm to 140 nm, 90 nm to 130 nm, 90 nm to 120 nm, 90 nm to 110 nm, 90 nm to 100 nm, 100 nm to 300 nm, 110 nm to 290 nm, 120 nm to 280 nm, 130 nm to 270 nm, 140 nm to 260 nm, 150 nm to 250 nm, 160 nm to 240 nm, 170 nm to 230 nm, 180 nm to 220 nm, or 190 nm to 210 nm. The size of the EVs (e.g., exosomes) described herein can be measured according to the methods described below.

[0305] In some aspects, the EVs (e.g., exosomes) of the present disclosure comprise a bilayer lipid membrane (“EV (e.g., exosome) membrane”), which includes an inner surface and an outer surface. In certain embodiments, the inner surface faces the core (i.e., lumen) of the EV (e.g., exosome). In certain aspects, the outer surface can contact the endosome, multivesicular body, or membrane / cytoplasm of the producer cell or target cell.

[0306] In some aspects, the EV (e.g., exosome) membrane comprises lipids and fatty acids. In some aspects, the EV (e.g., exosome) membrane comprises phospholipids, glycolipids, fatty acids, sphingolipids, glycerophospholipids, sterols, cholesterol, and phosphatidylserine.

[0307] In some aspects, the EV (e.g., exosome) membrane comprises an inner leaflet and an outer leaflet. The composition of the inner leaflet and the outer leaflet can be determined by transbilayer distribution assays known in the art, see, e.g., Kuypers et al., Biochim Biophys Acta 1985 819:170. In some aspects, the composition of the outer leaflet is about 70 - 90% choline phospholipids, about 0 - 15% acidic phospholipids, and about 5 - 30% phosphatidylethanolamine. In some aspects, the composition of the outer leaflet is about 15 - 40% choline phospholipids, about 10 - 50% acidic phospholipids, and about 30 - 60% phosphatidylethanolamine.

[0308] On the one hand, the present disclosure relates to the generation and use of lumen-engineered EVs (e.g., exosomes). The lumen-engineered exosomes have an interior space (the luminal surface of the EV) that is modified in its composition, e.g., relative to the composition of exosomes that exist in nature. For example, the composition of the luminal surface can be modified by altering the protein, lipid, or glycan content of the components on the luminal side of the EV (e.g., exosome) membrane. In some aspects, the luminal surface of the EV (e.g., exosome) can comprise one or more recombinantly expressed proteins that are not native to the EV (e.g., exosome), such as the scaffold proteins of the present disclosure, e.g., exosome luminal proteins. Accordingly, the present disclosure provides compositions that allow for the anchoring of payloads (e.g., bioactive molecules) to EVs (e.g., exosomes), the anchoring comprising linking the payload (e.g., bioactive molecule) to a scaffold protein of the present disclosure (e.g., exosome luminal protein). The present disclosure also provides methods for anchoring a payload (e.g., bioactive molecule) to an EV (e.g., exosome), which comprise linking the bioactive molecule to a scaffold protein of the present disclosure (e.g., exosome luminal protein).

[0309] In some aspects, the scaffold proteins of the present disclosure (e.g., exosome luminal proteins) are attached to the luminal surface of the EV (e.g., exosome) by lipidation. In some aspects, the scaffold proteins of the present disclosure are fatty acylated. In some aspects, the scaffold proteins are myristoylated.

[0310] Myristoylation is a lipidation modification in which the myristoyl group derived from myristic acid is covalently attached to the α-amino group of the N-terminal glycine via an amide bond. Myristic acid is a 14-carbon saturated fatty acid (14:4), with the systematic name n-tetradecanoic acid. This modification can be added during or after translation. During co-translational addition of the myristoyl group, the N-terminal methionine residue in the newly formed, growing polypeptide is cleaved and the N-terminal glycine is modified. This occurs in approximately 80% of myristoylated proteins. Post-translational myristoylation typically occurs after a caspase cleavage event, resulting in the exposure of an internal glycine residue, which can then be used for the addition of myristic acid. In addition to co-translational or post-translational myristoylation (in vivo or in vitro, e.g., by an enzyme), myristoylation of the scaffold proteins of the present disclosure, such as exosome luminal proteins, can also occur by chemical synthesis (e.g., by attaching myristic acid to the scaffold protein as a synthetic step during chemical synthesis).

[0311] In some embodiments, lipid anchoring to the biological membrane is not palmitoylation (i.e., the attachment of palmitic acid, typically to cysteine, and less frequently to serine or threonine). In other embodiments, lipid anchoring to the biological membrane is prenylation (attachment of isoprenoids) or glycosylphosphatidylinositol linkage (GPI-linkage). Prenylated proteins are proteins that have a covalently attached hydrophobic isoprenoid polymer (i.e., branched five-carbon hydrocarbon) at a cysteine residue of the protein. GPI-linked proteins are attached to a group of the GPI complex molecule by an amide bond to the C-terminal carboxyl group of the protein. GPI attachment occurs through the action of a GPI-transamidase complex. The fatty acid chain of phosphatidylinositol inserts into the membrane and is thus the reason for anchoring the protein to the membrane.

[0312] In some embodiments, lumen-engineered EVs (e.g., exosomes) are produced by chemical and / or physical methods such as PEG-induced fusion and / or sonication fusion.

[0313] In other embodiments, lumen-engineered EVs (e.g., exosomes) are produced by genetic engineering. Exosomes produced from genetically modified producer cells or the progeny of genetically modified cells may contain a modified lumen composition. In some embodiments, lumen-engineered EVs (e.g., exosomes) have EV (e.g., exosome) proteins (e.g., scaffolding proteins, e.g., exosome lumen proteins such as MARCKS, MARKSL1, BASP1, or a combination thereof) at a higher or lower density, or contain modifications or fragments of EV (e.g., exosome) proteins (e.g., any functional fragment, variant, or derivative thereof, or any combination thereof).

[0314] For example, lumen-engineered EVs (e.g., exosomes) can be produced from cells transformed with an exogenous sequence encoding an EV (e.g., exosome) protein or a modification or fragment of an EV (e.g., exosome) protein (e.g., a scaffolding protein, e.g., an exosome lumen protein such as MARCKS, MARKSL1, BASP1, any functional fragment, variant, or derivative thereof, or any combination thereof). EVs (e.g., exosomes) (including proteins expressed from the exogenous sequence) can include a modified lumen surface protein (scaffolding protein) composition.

[0315] Various modifications or fragments of EV proteins (e.g., exosome proteins), such as scaffolding proteins (e.g., exosome lumen proteins such as MARCKS, MARKSL1, BASP1, any functional fragment, variant or derivative thereof, or any combination thereof) can be used in the embodiments of the present disclosure. For example, proteins modified to more effectively target the lumen surface of EVs (e.g., exosomes) can be used. Proteins modified to contain the minimal fragment required for specific and effective targeting of the lumen surface of EVs (e.g., exosomes) can also be used.

[0316] In some aspects, the scaffolding proteins of the present disclosure, such as exosome lumen proteins, include MARCKS protein or its fragments, variants or derivatives. The MARCKS protein (Uniprot accession number P29966) is also known as protein kinase C substrate, 80 kDa protein, light chain. The full-length human MARCKS protein is 332 amino acids in length and contains a calmodulin-binding domain at amino acid residues 152 - 176. In some embodiments, the scaffolding proteins of the present disclosure include the mature MARCKS protein (i.e., without the N-terminal methionine). In some aspects, the scaffolding proteins of the present disclosure are derived from the mature MARCKS protein, i.e., they are fragments, variants or derivatives of the mature MARCKS protein and thus lack the N-terminal methionine present in the immature protein.

[0317] In some aspects, the scaffolding proteins of the present disclosure, such as exosome lumen proteins, include MARCKSL1 protein (Uniprot accession number P49006) (also known as MARCKS-like protein 1) and macrophage myristoylated alanine-rich C kinase substrate. The full-length human MARCKSL1 protein is 195 amino acids in length. The MARCKSL1 protein has an effector domain involved in lipid binding and calmodulin binding at amino acid residues 87 - 110. In some embodiments, the scaffolding proteins of the present disclosure include the mature MARCKSL1 protein (i.e., without the N-terminal methionine). In some aspects, the scaffolding proteins of the present disclosure are derived from the mature MARCKSL1 protein, i.e., they are fragments, variants or derivatives of the mature MARCKSL1 protein and thus lack the N-terminal methionine present in the immature protein.

[0318] In some aspects, the scaffolds of the present disclosure include BASP1 protein (Uniprot accession number P80723), also known as 22 kDa neuron tissue-enriched acidic protein or neuronal axonal membrane protein NAP-22. The full-length human BASP1 protein sequence (isoform 1) is 227 amino acids in length. The isoform generated by alternative splicing lacks amino acids 88 to 141 from isoform 1. In some embodiments, the scaffold proteins of the present disclosure, such as exosome lumen proteins, include the mature BASP1 protein (i.e., without the N-terminal methionine). In some aspects, the scaffold proteins of the present disclosure are derived from the mature BASP1 protein, i.e., they are fragments, variants, or derivatives of the mature BASP1 protein, and thus lack the N-terminal methionine present in the immature protein.

[0319] In some aspects, the scaffold proteins of the present disclosure, such as exosome lumen proteins, comprise an "N-terminal domain" (ND) and an "effector domain", wherein the ND and / or ED associates with the inner lumen surface of an EV (e.g., an exosome). As used herein, the term "associates with" refers to the interaction between the scaffold protein of the present disclosure and the inner lumen surface of an EV (e.g., an exosome), which does not involve covalent linkage to membrane components. For example, the scaffolds of the present disclosure can associate with the inner lumen surface of an EV, for example, via a lipid anchor (e.g., myristic acid) and / or a multi-domain electrostatically interacting with the negatively charged head of membrane phospholipids. In other aspects, the scaffold protein comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND associates with the inner lumen surface of the EV, and the ED associates with the inner lumen surface of the EV via ionic interactions, wherein the ED contains at least two, at least three, at least four, at least five, at least six, or at least seven consecutive lysines (Lys) in the sequence.

[0320] In other aspects, the ED further comprises one or more low-complexity regions, such as PEST motifs. A PEST sequence is a peptide sequence rich in proline (P), glutamate (E), serine (S), and threonine (T). In some aspects, the ED further comprises negatively charged residues (e.g., Glu) as well as a number of Ser and Thr, which undergo transient phosphorylation (thus adding negative charges to the region outside the ED).

[0321] In some aspects, the ND associates with the inner lumen surface of an EV (e.g., an exosome) by lipidation, such as by myristoylation. In some embodiments, the ND has a Gly at the N-terminus. In some embodiments, the N-terminal Gly is myristoylated. In some embodiments, the ND does not contain a Met at the N-terminus. In other embodiments, the ND contains a myristoylated Gly and does not contain a Met at the N-terminus.

[0322] In some aspects, ED associates with the luminal surface of EVs (e.g., exosomes) through ionic interactions. In some embodiments, ED associates with the luminal surface of EVs (e.g., exosomes) through electrostatic interactions, particularly attractive electrostatic interactions.

[0323] In some aspects, ED comprises (i) a basic amino acid (e.g., lysine), or (ii) two or more basic amino acids (e.g., lysine) adjacent to each other in the polypeptide sequence. In some aspects, the basic amino acid is lysine (Lys; K), arginine (Arg, R), or histidine (His, H). In some embodiments, the basic amino acid is (Lys)n, where n is an integer between 1 and 10.

[0324] In other aspects, if the N-terminus of ED is directly linked to the lysine at the C-terminus of ND, i.e., the lysine is in the N-terminus of ED and is fused to the lysine in the C-terminus of ND, then ED comprises at least lysine and ND comprises lysine at the C-terminus. In other embodiments, when the N-terminus of ED is linked to the C-terminus of ND through a linker (e.g., one or more amino acids), ED comprises at least two lysines, at least three lysines, at least four lysines, at least five lysines, at least six lysines, or at least seven lysines.

[0325] In some aspects, ED comprises K, KK, KKK, KKKK (SEQ ID NO:151), KKKKK (SEQ ID NO:152), or any combination thereof. The present disclosure also provides that, in some aspects, lysine repeats can be replaced by arginine. In other aspects, arginine repeats in ED or the scaffold protein provide lower loading efficacy compared to lysine efficacy.

[0326] In some aspects, the scaffolding proteins useful in the present disclosure, such as exophilin lumen proteins, require at least two lysines or at least three lysines, which are repeated in sequence in the ED or in the ED and ND, i.e., the lysines are at the C-terminus of the ND and the Ks are in the N-terminus of the ED. In some embodiments, the ED contains K, KK, KKK, KKKK (SEQ ID NO: 151), KKKKK (SEQ ID NO: 152), or any combination thereof. In some aspects, the ND contains an amino acid sequence as shown by G:X2:X3:X4:X5:X6, where G represents Gly; where ":" represents a peptide bond, where each of X2 to X6 independently represents an amino acid; and where X6 represents a basic amino acid. In some aspects, the X6 amino acid is selected from the group consisting of Lys, Arg, and His. In some aspects, the X5 amino acid is selected from the group consisting of Pro, Gly, Ala, and Ser. In some aspects, the X2 amino acid is selected from the group consisting of Pro, Gly, Ala, and Ser. In some aspects, X4 is selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Gln, and Met. In some aspects, the scaffolding protein does not contain a Met (e.g., myristoylated) at the N-terminus.

[0327] In some aspects, the scaffolding protein, such as exophilin lumen protein, contains an ND and an ED, where the ND contains an amino acid sequence as shown by G:X2:X3:X4:X5:X6, where G represents Gly; where ":" represents a peptide bond, where each of X2 to X6 independently represents an amino acid; where X6 represents a basic amino acid, the X5 amino acid is selected from the group consisting of Pro, Gly, Ala, and Ser, X4 is selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Gln, and Met, X3 represents an amino acid, and the X2 amino acid is selected from the group consisting of Pro, Gly, Ala, and Ser, where the ED contains at least one amino acid, e.g., at least one Lys, and where the scaffolding protein is not (i) an amino acid sequence containing SEQ ID NO: 4-114, 116-118, 122-150, or 180-190, or (ii) an amino acid sequence encoded by SEQ ID NO: 115 or 118 to 121.

[0328] In some aspects, a scaffolding protein, such as an exomer lumen protein, comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND comprises an amino acid sequence as shown by G:X2:X3:X4:X5:X6, wherein G is glycine represented as Gly; wherein ":" represents a peptide bond; wherein each of X2 to X6 is independently an amino acid; wherein X6 comprises a basic amino acid; and wherein the ED is linked to X6 by a peptide bond and comprises at least one lysine at the N-terminus of the ED. In some aspects, the scaffolding protein does not comprise Met (e.g., myristoylated) at the N-terminus. In other aspects, the scaffolding protein does not comprise or consist of the following amino acid sequences: (i) an amino acid sequence containing SEQ ID NO:4-114, 116-118, 122-150 or 180-190, or (ii) an amino acid sequence encoded by SEQ ID NO:115 or 118 to 121.

[0329] In some embodiments, the ND of a scaffolding protein, such as an exomer lumen protein, comprises the amino acid sequence G:X2:X3:X4:X5:X6, wherein

[0330] a. G represents Gly;

[0331] b. ":" represents a peptide bond;

[0332] c. X2 represents an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser;

[0333] d. X3 represents any amino acid;

[0334] e. X4 represents an amino acid selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Gln, and Met;

[0335] f. X5 represents an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser; and

[0336] g. X6 represents an amino acid selected from the group consisting of Lys, Arg, and His.

[0337] In some aspects, scaffolding proteins such as exocyst lumen proteins do not contain a Met at the N-terminus (e.g., myristoylated). In other aspects, the scaffolding protein does not comprise or consist of the following amino acid sequences: (i) an amino acid sequence comprising SEQ ID NO: 4 to 114, 116 to 118, 122 to 150, or 180 to 190 or (ii) an amino acid sequence encoded by SEQ ID NO: 115 or 118 to 121. In some aspects, the X3 amino acid is selected from the group consisting of Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, and Arg.

[0338] In some aspects, ND and ED are linked by a linker. In some embodiments, the linker comprises one or more amino acids. In some aspects, the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some aspects, two or more linkers can be connected in series. Generally, the linker provides flexibility or prevents / reduces steric hindrance. The linker is generally not cleaved; however, in certain aspects, such cleavage may be desirable. Thus, in some embodiments, the linker may comprise one or more protease-cleavable sites, which may be located within the linker sequence or flanking the linker at either end of the linker sequence. When ND and ED are linked by a linker, ED contains at least two lysines, at least three lysines, at least four lysines, at least five lysines, at least six lysines, or at least seven lysines.

[0339] In some aspects, the linker is a peptide linker. In some aspects, the peptide linker may comprise at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids.

[0340] In some aspects, the linker is a glycine / serine linker. In some embodiments, the peptide linker is a glycine / serine linker according to the formula [(Gly)n-Ser]m, where n is any integer from 1 to 100, and m is any integer from 1 to 100. In other aspects, the glycine / serine linker is according to the formula [(Gly)x-Sery]z, where x is an integer from 1 to 4, y is 0 or 1, and z is an integer from 1 to 50. In some aspects, the peptide linker comprises the sequence Gn, where n can be an integer from 1 to 100. In some aspects, the peptide linker can comprise the sequence (GlyAla)n, where n is an integer from 1 to 100. In other aspects, the peptide linker can comprise the sequence (GlyGlySer)n, where n is an integer between 1 and 100.

[0341] In some aspects, the peptide linker is synthetic, i.e., not naturally occurring. In one embodiment, the peptide linker comprises a peptide (or polypeptide) (e.g., a natural or non-naturally occurring peptide) that contains an amino acid sequence that links or genetically fuses a first linear amino acid sequence to a second linear amino acid sequence, which is not naturally linked or genetically fused to the second linear amino acid sequence. For example, in one aspect, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., containing mutations such as additions, substitutions, or deletions).

[0342] In other aspects, the peptide linker can comprise non-naturally occurring amino acids. In still other aspects, the peptide linker can comprise naturally occurring amino acids in a linear sequence that does not exist in nature. In other aspects, the peptide linker can comprise a naturally occurring polypeptide sequence.

[0343] The present disclosure also provides isolated extracellular vesicles (EVs) (e.g., exosomes) that contain a bioactive molecule linked to a scaffold protein such as exosomal lumen protein, where the scaffold protein comprises ND-ED, wherein:

[0344] a. ND comprises G:X2:X3:X4:X5:X6; wherein:

[0345] i. G represents Gly;

[0346] ii. ":" represents a peptide bond;

[0347] iii. X2 represents an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser.

[0348] iv. X3 represents any amino acid;

[0349] v. X4 represents an amino acid selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Glu, and Met;

[0350] vi. X5 represents an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser.

[0351] vii. X6 represents an amino acid selected from the group consisting of Lys, Arg, and His;

[0352] b. “—” represents an optional linker; and

[0353] c. ED is an effector domain that contains (i) at least two consecutive lysines (Lys), where the N-terminal lysine is linked to X6 by a peptide bond or one or more amino acids; or (ii) at least one lysine that is directly linked to X6 by a peptide bond. In some aspects, the scaffold protein does not contain a Met (e.g., myristoylated) at the N-terminus. In other aspects, the scaffold protein does not contain or is not composed of the following amino acid sequences: (i) an amino acid sequence containing SEQ ID NO: 4 to 114, 116 to 118, 122 to 150, or 180 to 190; or (ii) an amino acid sequence encoded by SEQ ID NO: 115 or 118 to 121.

[0354] In some aspects, the X2 amino acid is selected from the group consisting of Gly and Ala. In some aspects, the X3 amino acid is Lys. In some aspects, the X4 amino acid is Leu or Glu. In some aspects, the X5 amino acid is selected from the group consisting of Ser and Ala. In some aspects, the X6 amino acid is Lys. In other embodiments, the X2 amino acid is Gly, Ala, or Ser; the X3 amino acid is Lys or Glu, the X4 amino acid is Leu, Phe, Ser, and Glu, the X5 amino acid is Ser or Ala; and the X6 amino acid is Lys. In some aspects, the — linker contains a peptide bond or one or more amino acids.

[0355] In some aspects, the ED in the scaffold protein, such as the endosome lumen protein, contains Lys (K), KK, KKK, KKKK (SEQ ID NO: 151), KKKKK (SEQ ID NO: 152), Arg (R), RR, RRR, RRRR (SEQ ID NO: 153); RRRRR (SEQ ID NO: 154), KR, RK, KKR, KRK, RKK, KRR, RRK, (K / R)(K / R)(K / R)(K / R) (SEQ ID NO: 155), (K / R)(K / R)(K / R)(K / R)(K / R) (SEQ ID NO: 156), or any combination thereof.

[0356] In some aspects, scaffolding proteins such as exomer lumen proteins contain amino acid sequences such as (i) GGKLSKK (SEQ ID NO:157), (ii) GAKLSKK (SEQ ID NO:158), (iii) GGKQSKK (SEQ ID NO:159), (iv) GGKLAKK (SEQ ID NO:160), or (v) any combination thereof. In some aspects, the scaffolding protein does not contain an amino acid sequence having a Met at the N-terminus of the sequence in (i) to (v). In other aspects, the scaffolding protein does not contain or consist of the following amino acid sequences: (i) an amino acid sequence comprising SEQ ID NO:4 to 114, 116 to 118, or 122 to 150, or (ii) an amino acid sequence encoded by SEQ ID NO:115 or 118 to 121.

[0357] In some aspects, the ND in a scaffolding protein such as an endophilin contains the following amino acid sequences: (i) GGKLSK (SEQ ID NO: 203), (ii) GAKLSK (SEQ ID NO: 204), (iii) GGKQSK (SEQ ID NO: 205), (iv) GGKLAK (SEQ ID NO: 206), or (v) any combination thereof, and the ED in the scaffolding protein contains (a) K, (b) KK, (c) KKK, (d) KKKG (SEQ ID NO: 207), (e) KKKGY (SEQ ID NO: 208), (f) KKKGYN (SEQ ID NO: 209), (g) KKKGYNV (SEQ ID NO: 210), (h) KKKGYNVN (SEQ ID NO: 211), (i) KKKGYS (SEQ ID NO: 212), (k) KKKGYG (SEQ ID NO: 213), (l) KKKGYGG (SEQ ID NO: 214), (m) KKKGS (SEQ ID NO: 215), (n) KKKGSG (SEQ ID NO: 216), (o) KKKGSG (SEQ ID NO: 217), (p) KKKGSGS (SEQ ID NO: 218), (q) KKKS (SEQ ID NO: 219), (r) KKKSG (SEQ ID NO: 220), (s) KKKSGG (SEQ ID NO: 221), (t) KKKSGGS (SEQ ID NO: 222), (u) KKKSGGSG (SEQ ID NO: 223), (v) KKSGGSGG (SEQ ID NO: 224), (w) KKKSGGSGGS (SEQ ID NO: 225), (x) KRFSFKKS (SEQ ID NO: 226), or any combination thereof. In some aspects, the scaffolding protein does not contain an amino acid sequence having a Met at the N-terminus of the sequence in (i) to (v). In some aspects, the scaffolding protein does not contain or consist of the following amino acid sequences: (i) an amino acid sequence containing SEQ ID NO: 4 to 114, 116 to 118, 122 to 150, or 180 to 190, or (ii) an amino acid sequence encoded by SEQ ID NO: 115 or 118 to 121.

[0358] In some aspects, the polypeptide sequence of a scaffold protein such as an exosome lumen protein consists of an amino acid sequence selected from the group consisting of: (i) GGKLSKK (SEQ ID NO: 157), (ii) GAKLSKK (SEQ ID NO: 158), (iii) GGKQSKK (SEQ ID NO: 159), (iv) GGKLAKK (SEQ ID NO: 160), or (v) any combination thereof.

[0359] In some aspects, a scaffold protein such as an exosome lumen protein comprises the following amino acid sequences: (i) GGKLSKKK (SEQ ID NO: 161), (ii) GGKLSKKS (SEQ ID NO: 162), (iii) GAKLSKKK (SEQ ID NO: 163), (iv) GAKLSKKS (SEQ ID NO: 164), (v) GGKQSKKK (SEQ ID NO: 165), (vi) GGKQSKKS (SEQ ID NO: 166), (vii) GGKLAKKK (SEQ ID NO: 167), (viii) GGKLAKKS (SEQ ID NO: 168), or (ix) any combination thereof. In some aspects, the scaffold protein does not comprise an amino acid sequence having Met at the N-terminus of the sequences in (i) to (ix). In some aspects, the scaffold protein does not comprise or consist of the following amino acid sequences: (i) an amino acid sequence comprising SEQ ID NO: 4 to 114, 116 to 118, 122 to 150, or 180 to 190, or (ii) an amino acid sequence encoded by SEQ ID NO: 115 or 118 to 121.

[0360] In some aspects, the polypeptide sequence of a scaffold protein such as an exosome lumen protein of the present disclosure consists of an amino acid sequence selected from the group consisting of: (i) GGKLSKKK (SEQ ID NO: 161), (ii) GGKLSKKS (SEQ ID NO: 162), (iii) GAKLSKKK (SEQ ID NO: 163), (iv) GAKLSKKS (SEQ ID NO: 164), (v) GGKQSKKK (SEQ ID NO: 165), (vi) GGKQSKKS (SEQ ID NO: 166), (vii) GGKLAKKK (SEQ ID NO: 167), (viii) GGKLAKKS (SEQ ID NO: 168), and (ix) any combination thereof.

[0361] In some aspects, the length of a scaffolding protein such as an exomer lumen protein is at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 50, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least 85, at least about 90, at least about 95, at least about 100, at least about 105, at least about 110, at least about 115, at least about 120, at least about 125, at least about 130, at least about 135, at least about 140, at least about 145, at least about 150, at least about 155, at least about 160, at least about 165, at least about 170, at least about 175, at least about 180, at least about 185, at least about 190, at least about 195, at least about 200, at least about 205, at least about 210, at least about 215, at least about 220, at least about 225, at least about 230, at least about 235, at least about 240, at least about 245, at least about 250, at least about 255, at least about 260, at least about 265, at least about 270, at least about 275, at least about 280, at least about 285, at least about 290, at least about 295, at least about 300, at least about 305, at least about 310, at least about 315, at least about 320, at least about 325, at least about 330, at least about 335, at least about 340, at least about 345, or at least about 350 amino acids.

[0362] In some aspects, the length of a scaffolding protein such as an exomer lumen protein is from about 5 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150, about 150 to about 160, about 160 to about 170, about 170 to about 180, about 180 to about 190, about 190 to about 200, about 200 to about 210, about 210 to about 220, about 220 to about 230, about 230 to about 240, about 240 to about 250, about 250 to about 260, about 260 to about 270, about 270 to about 280, about 280 to about 290, about 290 to about 300, about 300 to about 310, about 310 to about 320, about 320 to about 330, about 330 to about 340 or about 340 to about 250 amino acids.

[0363] In some aspects, scaffolding proteins such as exomer lumen proteins comprise (i) GGKLSKKKKGYNVN (SEQ ID NO:169), (ii) GAKLSKKKKGYNVN (SEQ ID NO:170), (iii) GGKQSKKKKGYNVN (SEQ ID NO:171), (iv) GGKLAKKKKGYNVN (SEQ ID NO:172), (v) GGKLSKKKKGYSGG (SEQ ID NO:173), (vi) GGKLSKKKKGSGGS (SEQ ID NO:174), (vii) GGKLSKKKKSGGSG (SEQ ID NO:175), (viii) GGKLSKKKSGGSGG (SEQ ID NO:176), (ix) GGKLSKKSGGSGGS (SEQ ID NO:177), (x) GGKLSKSGGSGGSV (SEQ ID NO:178), or (xi) GAKKSKKRFSFKKS (SEQ ID NO:179). In some aspects, the scaffolding protein does not comprise an amino acid sequence having a Met at the N-terminus of the sequence in (i) to (xi). In some aspects, the scaffolding protein does not comprise or consist of the following amino acid sequences: (i) an amino acid sequence comprising SEQ ID NO:4 to 114, 116 to 118, 122 to 150, or 180 to 190, or (ii) an amino acid sequence encoded by SEQ ID NO:115 or 118 to 121.

[0364] In some aspects, the polypeptide sequences of the scaffold proteins of the present disclosure, such as the foreign body lumen protein, consist of: (i) GGKLSKKKKGYNVN (SEQ ID NO: 169), (ii) GAKLSKKKKGYNVN (SEQ ID NO: 170), (iii) GGKQSKKKKGYNVN (SEQ ID NO: 171), (iv) GGKLAKKKKGYNVN (SEQ ID NO: 172), (v) GGKLSKKKKGYSGG (SEQ ID NO: 173), (vi) GGKLSKKKKGSGGS (SEQ ID NO: 174), (vii) GGKLSKKKKSGGSG (SEQ ID NO: 175), (viii) GGKLSKKKSGGSGG (SEQ ID NO: 176), (ix) GGKLSKKSGGSGGS (SEQ ID NO: 177), (x) GGKLSKSGGSGGSV (SEQ ID NO: 178), or (xi) GAKKSKKRFSFKKS (SEQ ID NO: 179). In some aspects, the scaffold protein does not contain an amino acid sequence with a Met at the N-terminus having the sequences in (i) to (xi). In some aspects, the scaffold protein does not contain or consist of the following amino acid sequences: (i) an amino acid sequence containing SEQ ID NO: 4 to 114, 116 to 118, 122 to 150, or 180 to 190, or (ii) an amino acid sequence encoded by SEQ ID NO: 115 or 118 to 121.

[0365] In other aspects, the scaffold protein, such as the foreign body lumen protein, contains any of the sequences disclosed herein, but does not contain the corresponding sequence with a Met at the N-terminus, e.g., SEQ ID NO: 4 to 109. In some aspects, the scaffold protein contains any of the sequences in Table 1, but does not contain or consist of the following amino acid sequences: (i) an amino acid sequence containing SEQ ID NO: 4 to 114, 116 to 118, 122 to 150, or 180 to 190, or (ii) an amino acid sequence encoded by SEQ ID NO: 115 or 118 to 121.

[0366] The following are non-limiting examples of scaffold proteins that can be used in the present disclosure, such as foreign body lumen proteins:

[0367] Table 1

[0368]

[0369]

[0370]

[0371]

[0372] In some aspects, the scaffold proteins of the present disclosure, such as exophilin lumen proteins, consist of or consist essentially of any of the sequences in the present disclosure. In some aspects, the scaffolds of the present disclosure consist of the sequences disclosed in Table 1. In some aspects, the scaffolds of the present disclosure consist of sequences disclosed herein (such as the sequences disclosed in Table 1) covalently attached to a membrane anchor (e.g., myristic acid). In some aspects, membrane anchoring can be achieved by lipidation. In some embodiments, the lipidation can be, for example, fatty acylation. In some aspects, the fatty acylation can be, for example, myristoylation. In some aspects, the membrane anchor is covalently attached to the N-terminal amino acid of a sequence disclosed herein, such as the sequences disclosed in Table 1. In some aspects, the membrane anchor is covalently attached to the N-terminal glycine of a sequence disclosed herein, such as the sequences disclosed in Table 1. Thus, in some aspects, the scaffolds of the present disclosure consist of disclosed sequences that have been N-myristoylated, such as the sequences disclosed in Table 1.

[0373] In some aspects, the scaffold proteins of the present disclosure, such as exophilin lumen proteins, do not contain an N-terminal Met. In some aspects, the scaffold protein contains a lipidated amino acid, such as a myristoylated amino acid, at the N-terminus of the scaffold protein, which serves as a lipid anchor. In some aspects, the amino acid residue at the N-terminus of the scaffold protein is Gly. The presence of N-terminal Gly is required for N-myristoylation. In some aspects, the amino acid residue at the N-terminus of the scaffold protein is synthetic. In some aspects, the amino acid residue at the N-terminus of the scaffold protein is a glycine analogue, such as allylglycine, butylglycine, or propargylglycine.

[0374] In some aspects, the lipid anchor can be attached to any N-terminal amino acid of the scaffold proteins of the present disclosure either chemically or enzymatically.

[0375] In other aspects, the lipid anchor can be any lipid anchor known in the art, such as palmitic acid or glycosylphosphatidylinositol. In rare cases, for example, by using a myristic acid-restricted medium, some other fatty acids (including short-chain and unsaturated fatty acids) can be attached to the N-terminal glycine. For example, it has been reported that in BK channels, myristate is attached post-translationally to internal serine / threonine or tyrosine residues via a hydroxyester bond. Membrane anchors known in the art are given in the following table.

[0376] Table 2

[0377]

[0378] In some embodiments, a scaffold protein, such as an exosomal lumen protein, comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the mature form of SEQ ID NO:1 (MARCKS), SEQ ID NO:2 (MARCKSL1), or SEQ ID NO:3 (BASP1) (i.e., the N-terminal methionine is absent in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3).

[0379] In some embodiments, a scaffold protein (e.g., an exosomal lumen protein) comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to a functional fragment of the mature form of SEQ ID NO:1 (MARCKS), SEQ ID NO:2 (MARCKSL1), or SEQ ID NO:3 (BASP1) (i.e., the N-terminal methionine amino acid is absent in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3).

[0380] In some aspects, a bioactive molecule attached to the scaffold protein of the present disclosure is on the luminal side of an EV (e.g., an exosome).

[0381] In some aspects, a scaffold protein, such as an exosomal lumen protein, further comprises a transmembrane domain. In some aspects, the transmembrane domain is inserted between the ED domain of the scaffold protein located on the luminal side of the EV (e.g., an exosome) and the payload (e.g., a bioactive molecule). Thus, the payload (e.g., a bioactive molecule) is anchored to the outer surface of the EV (e.g., an exosome).

[0382] In some aspects, a scaffold protein, such as an exosomal lumen protein, further comprises an extravesicular domain. In some embodiments, the extravesicular domain is inserted between the transmembrane domain and the bioactive molecule.

[0383] In some aspects, the scaffold protein is linked to the bioactive molecule by at least one linker, e.g., a peptide linker. In some aspects, ND is linked to ED by a linker inserted directly between the two domains. In some aspects, the linker comprises, for example, a peptide bond or one or more amino acids. In some aspects, the linker includes a cleavable linker. In some aspects, the linker includes a flexible linker. In some aspects, the linker includes a self-immolative linker.

[0384] Fusions between the scaffold proteins of the present disclosure and bioactive molecules (e.g., molecules having therapeutic activity) can also be used. For example, the fusion protein can comprise a scaffold protein such as MARCKS, MARCKSL1, BASP1, etc. or modifications thereof, particularly fragments or variants thereof, and a payload such as a bioactive molecule (e.g., a therapeutic peptide). In some aspects, the fusion protein comprises a scaffold protein containing a fragment with the amino terminus of BASP1. In other aspects, the bioactive molecules include proteins, polypeptides, peptides, polynucleotides (DNA and / or RNA), compounds, viruses, ionophores, carriers of ionophores, moieties forming channels or pores, or any combination thereof.

[0385] The bioactive molecule (e.g., a therapeutic peptide) can be selected from the group consisting of: natural peptides, recombinant peptides, synthetic peptides, fusion proteins, or linkers conjugated to therapeutic compounds. The bioactive molecule (e.g., a therapeutic compound) can also be a nucleotide, an amino acid, a lipid, a carbohydrate, or a small molecule. The bioactive molecule (e.g., a therapeutic peptide) can be an antibody, an enzyme, a ligand, an antigen (e.g., a tumor antigen or an antigen from an infectious agent such as a bacterium, virus, fungus, or protozoan), a receptor, an antimicrobial peptide, a transcription factor, or a fragment or modification thereof.

[0386] A fusion protein comprising a scaffold of the present disclosure fused or conjugated to a payload such as a bioactive molecule can be attached to the luminal surface of an EV (e.g., an exosome) and provide, for example, therapeutic activity to the EV (e.g., an exosome).

[0387] In some embodiments, the bioactive molecule (e.g., a therapeutic peptide) is a component of a genome editing complex. In some embodiments, the genome editing complex is a transcription activator-like effector nuclease (TAL-effector nuclease or TALEN); a zinc finger nuclease (ZFN); a recombinase; a CRISPR / Cas9 complex, a CRISPR / Cpf1 complex, a CRISPR / C2c1, C2c2, or C2c3 complex, a CRISPR / CasY or CasX complex, or any other suitable CRISPR complex known in the art; or any other suitable genome editing complex known in the art or any combination thereof.

[0388] In some embodiments, the bioactive molecule (e.g., a therapeutic peptide) is a transmembrane peptide. The transmembrane peptides described herein can be expressed as a fusion protein with any sequence described herein or any fragment or variant thereof. In some embodiments, the transmembrane protein has a first terminus fused to a luminal sequence in the lumen of an EV (e.g., an exosome) and a second terminus expressed on the surface of the EV (e.g., an exosome).

[0389] In some embodiments, an EV of the present disclosure (e.g., ) may comprise a second scaffold protein. In some embodiments, the second scaffold protein comprises a transmembrane protein, and the transmembrane protein comprises a PTGFRN polypeptide, a BSG polypeptide, an IGSF2 polypeptide, an IGSF3 polypeptide, an IGSF8 polypeptide, an ITGB1 polypeptide, an ITGA4 polypeptide, an SLC3A2 polypeptide, an ATP transporter polypeptide, an aminopeptidase N (ANPEP) polypeptide, an ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1) polypeptide, an enkephalinase (MME) polypeptide, a neuropilin-1 (NRP1) polypeptide, or a fragment, variant, or derivative thereof. Non-limiting examples of the second scaffold protein can be found in U.S. Patent No. 10,195,290 and PCT Publication No. WO 2019 / 040920, which are incorporated herein by reference in their entirety.

[0390] In some embodiments, the bioactive molecule (e.g., therapeutic peptide) is a nucleic acid-binding protein. In some embodiments, the nucleic acid-binding protein is Dicer, an Argonaute protein, TRBP, an MS2 bacteriophage coat protein. In some embodiments, the nucleic acid-binding protein further comprises one or more RNA or DNA molecules. In some embodiments, the one or more RNA are miRNA, siRNA, guide RNA, lincRNA, mRNA, antisense RNA, dsRNA, or a combination thereof.

[0391] In some embodiments, the bioactive molecule (e.g., therapeutic peptide) is part of a protein-protein interaction system. In some embodiments, the protein-protein interaction system comprises an FRB-FKBP interaction system, e.g., the FRB-FKBP interaction system described in Banaszynski et al., J Am Chem Soc. April 6, 2005; 127(13):4715-21.

[0392] The fusion protein can be targeted to the luminal surface of an EV (e.g., exosome) and provide therapeutic activity to the EV (e.g., exosome).

[0393] In some embodiments, a fusion protein with a targeting moiety is used. For example, the fusion protein can comprise (i) a scaffold protein, such as MARCKS, MARCKSL1, BASP1, or a fragment, variant, or modification thereof, and (ii) a targeting moiety. The targeting moiety can be used to target an EV (e.g., exosome) to a specific organ, tissue, or cell for treatment using the EV (e.g., exosome). In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof.

[0394] Antibodies and their antigen-binding fragments include intact antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies and their fragments, and also include single-chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments, such as, for example, scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, minibodies, camelid antibodies, and antibody-related polypeptides.

[0395] Antibodies and their antigen-binding fragments also include bispecific antibodies and multispecific antibodies, provided that they exhibit the desired biological activity or function. The modular structure of antibodies has been exploited to generate over 60 different bispecific antibody formats. See Spiess et al. (2015) Molecular Immunology 67:95-106, which is incorporated herein by reference in its entirety. Thus, in some embodiments, the bispecific antibody formats are selected from crossMab, DAF (dual action Fab) (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, Knobs-in-holes common LC, Knobs-in-holes assembly, charge pair, Fab arm exchange, SEEDbody, triomab, LUZ-Y (heterobispecific antibody with a leucine zipper that induces heterodimerization of 2 HCs), Fcab, Kλ-body, orthogonal Fab, DVD-IgG (dual variable domain IgG), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG (four-in-one), nanobody, nanobody-HSA, BiTE (bispecific T cell engager), diabody, DART (dual affinity retargeting), TandAb (tandem antibody), scDiabody, scDiabody-CH3, Triple Body, minibody, microbody, TriBi microbody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-ScFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HC Ab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intracellular antibody, Dock and Locck, ImmTAC, HSAbody, scDiabody-HSA, tandem scFv-toxin, IgG-IgG, Cov-X-Body, and scFv1-PEG-scFV2. A bispecific antibody can be a monospecific antibody engineered for bispecificity by attaching additional antigen-binding units at the amino or carboxyl terminus of the light or heavy chain. Alternatives to these additional antigen-binding units include single domain antibodies (unpaired VL or VH), paired antibody variable domains (e.g., Fv or scFv), or engineered protein scaffolds.Many bispecific fragment forms lacking some or all of the constant domains of bispecific antibodies are known in the art.

[0396] In some embodiments, the bioactive molecule (e.g., a therapeutic peptide) is a fusion protein comprising a scaffold protein of the present disclosure and a viral protein. In some embodiments, the viral protein comprises a viral capsid, an envelope protein, or a combination thereof. In some embodiments, the fusion protein permits the assembly of intact viruses on the luminal surface of EVs (e.g., exosomes).

[0397] In some embodiments, the bioactive molecule is an inhibitor of a negative checkpoint regulator or an inhibitor of a binding partner of a negative checkpoint regulator. In some embodiments, the negative checkpoint regulator is selected from the group consisting of cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell activation V domain Ig inhibitor (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73.

[0398] In some embodiments, the bioactive molecule is an immunogenic protein. In some embodiments, the bioactive molecule is a toxin, a toxoid, or a non-toxic mutant of a toxin. In some embodiments, the toxin is diphtheria toxin. In some embodiments, the toxoid is tetanus toxoid. In some embodiments, the non-toxic mutant of diphtheria toxin is a non-toxic mutant of diphtheria toxin.

[0399] In some embodiments, the bioactive molecule is an activator of a co-stimulatory molecule or an activator of a binding partner of a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is a TNF receptor superfamily member selected from the group consisting of: CD120a, CD120b, CD18, OX40, CD40, Fas receptor, M68, CD27, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, RANK, OCIF, TWEAK receptor, TACI, BAFF receptor, ATAR, CD271, CD269, AITR, TROY, CD358, TRAMP, and XEDAR. In some embodiments, the activator of the co-stimulatory molecule is a TNF receptor superfamily member selected from the group consisting of: TNFα, TNF-C, OX40L, CD40L, FasL, LIGHT, TL1A, CD27L, Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand, and EDA-2. In some embodiments, the co-stimulatory molecule is a CD28 superfamily co-stimulatory molecule. In some embodiments, the CD28 superfamily co-stimulatory molecule is ICOS or CD28. In some embodiments, the activator of the co-stimulatory molecule is ICOSL, CD80, or CD86.

[0400] In some embodiments, the bioactive molecule is a cytokine selected from the group consisting of: IL-2, IL-7, IL-10, IL-12, and IL-15. In some embodiments, the bioactive molecule is a protein comprising a T cell receptor (TCR), a T cell co-receptor, a major histocompatibility complex (MHC), a human leukocyte antigen (HLA), or a derivative thereof. In some embodiments, the bioactive molecule is a protein comprising a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, alpha-folate receptor, CE7R, IL-3, cancer-testis antigen, MART-1 gp100, and TNF-related apoptosis-inducing ligand.

[0401] In some aspects, a fusion protein comprising a bioactive molecule of the present disclosure and a scaffolding protein (e.g., MARCKS, MARCKSL1, BASP1, any one of SEQ ID NOs: 4-109, the corresponding sequence without the N-terminal M, or any scaffolding protein sequence disclosed herein (e.g., in Table 1), or a modification thereof, particularly a fragment or variant thereof) results in enrichment of the bioactive molecule in EVs (e.g., exosomes) compared to the expression of the bioactive molecule lacking the scaffolding protein. In some aspects, a fusion protein comprising a bioactive molecule and a scaffolding protein of the present disclosure (e.g., MARCKS without the N-terminal M, MARCKSL1, BASP1, the amino acid sequences of SEQ ID NOs: 4 to 114, 116 to 118, 122 to 150, or 180 to 190 without the N-terminal M, the amino acid sequences encoded by SEQ ID NO: 115 or 118 to 121, or any scaffolding protein sequence without the N-terminal M disclosed herein (e.g., in Table 1)) results in enrichment of the bioactive molecule in EVs (e.g., exosomes) compared to the expression of the bioactive molecule lacking the scaffolding protein.

[0402] In some embodiments, the scaffold proteins useful for the present disclosure include any one of SEQ ID NOs: 4-109, the corresponding sequences without N-terminal M, or any sequence disclosed in Table 1. In some aspects, the scaffold proteins useful for the present disclosure include any one of SEQ ID NOs: 4-109, wherein the scaffold protein does not contain an N-terminal Met. In some aspects, the scaffold protein contains a sequence disclosed in Table 1, wherein the scaffold protein does not contain an N-terminal Met. In some aspects, the scaffold proteins useful for the present disclosure include MARCKS, MARCKSL1, BASP1 without N-terminal M, the amino acid sequences of SEQ ID NOs: 4 to 114, 116 to 118, 122 to 150 or 180 to 190 without N-terminal M, the amino acid sequences encoded by SEQ ID NO: 115 or 118 to 121, or any scaffold protein sequence without N-terminal M disclosed herein (e.g., in Table 1). In some aspects, the scaffold proteins useful for the present disclosure include MARCKS, MARCKSL1 or BASP1, wherein the scaffold protein does not contain an N-terminal Met. In some aspects, the scaffold protein contains the amino acid sequences of SEQ ID NOs: 4 to 114, 116 to 118, 122 to 150 or 180 to 190, wherein the scaffold protein does not contain an N-terminal Met. In some aspects, the scaffold protein contains the amino acid sequences encoded by SEQ ID NO: 115 or 118 to 121, wherein the scaffold protein does not contain an N-terminal Met. In other embodiments, the scaffold proteins useful for the present disclosure contain any sequence disclosed herein, but not any one of SEQ ID NOs: 4-109, the corresponding sequences without N-terminal M, or any sequence without N-terminal M disclosed in Table 1. In other embodiments, the scaffold proteins useful for the present disclosure contain any sequence disclosed herein, but not any one of SEQ ID NOs: 4-109.

[0403] In some embodiments, the fusion protein comprises a scaffold protein, the scaffold protein comprising a peptide having the sequence MGXKLSKKK, where X is alanine or any other amino acid (SEQ ID NO: 117); or a peptide having the sequence (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), where the N-terminal M is cleaved such that the resulting fusion protein comprises GXKLSKKK (SEQ ID NO: 425) or (G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+) linked to a bioactive molecule, where each position within the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+), and position 6 is neither (+) nor (Asp or Glu). In some embodiments, the scaffold protein useful in the present disclosure is not MGXKLSKKK, where X is alanine or any other amino acid (SEQ ID NO: 117) or is not a peptide having the sequence (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+). In some embodiments, the scaffold protein useful in the present disclosure does not comprise MGXKLSKKK, where X is alanine or any other amino acid (SEQ ID NO: 117) or does not comprise a peptide having the sequence (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+).

[0404] In some embodiments, the fusion protein comprises a scaffold protein, the scaffold protein comprising a peptide having the sequence (M)(G)(π)(X)(Φ / π)(π)(+)(+) or (G)(π)(X)(Φ / π)(π)(+)(+), where each position within the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu). In some embodiments, the fusion protein comprises a scaffold protein, the scaffold protein comprising a peptide having the sequence (G)(π)(X)(Φ / π)(π)(+)(+), where the scaffold protein does not comprise an N-terminal M (e.g., N-terminal methionine).

[0405] In some aspects, the fusion protein comprises a scaffold protein, the scaffold protein comprising a peptide having the sequence (G)(π)(X)(Φ / π)(π)(+)(+), where each position within the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu), where the sequence is not (M)(G)(π)(X)(Φ / π)(π)(+)(+). In some embodiments, the scaffold protein comprises a peptide having the sequence (G)(π)(X)(Φ / π)(π)(+)(+), where the scaffold protein does not comprise an N-terminal Met.

[0406] In some embodiments, conventional EV (e.g., exosome) proteins are selected from the list consisting of: CD9, CD63, CD81, PDGFR, GPI-anchored proteins, LAMP2, LAMP2B, and fragments, variants, or derivatives thereof.

[0407] In some embodiments, a fusion protein comprising MARCKS, MARCKSL1, BASP1, or any one of SEQ ID NOs: 4-109 is enriched in exosomes up to >2-fold, >4-fold, >8-fold, >16-fold, >25-fold, >50-fold, >100-fold, >200-fold, >500-fold, >750-fold, >1,000-fold, >2,000-fold, >5,000-fold, >7,500-fold, >10,000-fold compared to a fusion protein lacking any one of MARCKS, MARCKSL1, BASP1, SEQ ID NOs: 4-109 or compared to a fusion protein comprising conventional EV (e.g., exosome) proteins. In some aspects, a fusion protein comprising MARCKS lacking an N-terminal M, MARCKSL1 lacking an N-terminal M, BASP1 lacking an N-terminal M, any one of SEQ ID NOs: 4-109 lacking an N-terminal M, or any sequence lacking an N-terminal M disclosed herein (e.g., in Table 1) is enriched in exosomes up to >2-fold, >4-fold, >8-fold, >16-fold, >25-fold, >50-fold, >100-fold, >200-fold, >500-fold, >750-fold, >1,000-fold, >2,000-fold, >5,000-fold, >7,500-fold, >10,000-fold compared to a fusion protein lacking any one of MARCKS lacking an N-terminal M, MARCKSL1 lacking an N-terminal M, BASP1 lacking an N-terminal M, SEQ ID NOs: 4-109 lacking an N-terminal M, or any sequence lacking an N-terminal M disclosed herein (e.g., in Table 1) or compared to a fusion protein comprising conventional EV (e.g., exosome) proteins.

[0408] In some embodiments, a fusion protein comprising MARCKS, MARCKSL1, BASP1, any one of SEQ ID NOs: 4-109, the corresponding sequence lacking an N-terminal M, or any scaffolding protein sequence lacking an N-terminal M disclosed herein (e.g., Table 1) is enriched in exosomes at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, at least about 1,000-fold, at least about 1,500-fold, at least about 2,000-fold, at least about 2,500-fold, at least about 3,000-fold, at least about 3,500-fold, at least about 4,000-fold, at least about 4,500-fold, at least about 5,000-fold, at least about 5,500-fold, at least about 6,000-fold, at least about 6,500-fold, at least about 7,000-fold, at least about 7,500-fold, at least about 8,000-fold, at least about 8,500-fold, at least about 9,000-fold, at least about 9,500-fold, or at least about 10,000-fold higher than a fusion protein lacking MARCKS, MARCKSL1, BASP1, any one of SEQ ID NOs: 4-109, the corresponding sequence lacking an N-terminal M, or any scaffolding protein sequence lacking an N-terminal M disclosed herein (e.g., Table 1), or compared to a fusion protein comprising conventional EV (e.g., exosome) proteins.

[0409] In some embodiments, the protein sequence of any one of SEQ ID NOs: 1-109, the corresponding sequence lacking an N-terminal M, or any scaffolding protein sequence lacking an N-terminal M disclosed herein (e.g., Table 1) is sufficient to load EVs (e.g., exosomes) with the fusion protein.

[0410] In some embodiments, the density of an endolumenally engineered EV (e.g., exosome) comprising a fusion protein containing an exogenous sequence (e.g., a bioactive molecule) and a newly identified EV (e.g., exosome) luminal protein (scaffolding protein) disclosed herein is higher than that of a similarly engineered EV (e.g., exosome) that comprises an exogenous sequence conjugated to a conventional EV (e.g., exosome) protein known in the art (e.g., CD9, CD63, CD81, PDGFR, GPI-anchored proteins, galectin, LAMP2 and LAMP2B, fragments thereof, or peptides that bind thereto).

[0411] In some embodiments, the density of the fusion protein comprising the newly identified EV (e.g., exosome) lumen protein (scaffold protein) herein on the inner lumen surface of the EV (e.g., exosome) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold that of the fusion protein on the inner lumen surface of other EVs (e.g., exosomes) similarly modified with conventional EV (e.g., exosome) proteins.

[0412] In some embodiments, the density of the fusion protein comprising the newly identified EV (e.g., exosome) lumen protein (scaffold protein) herein on the inner lumen surface of the EV (e.g., exosome) is 2- to 4-fold, 4- to 8-fold, 8- to 16-fold, 16- to 32-fold, 32- to 64-fold, 64- to 100-fold, 100- to 200-fold, 200- to 400-fold, 400- to 800-fold, 800- to 1,000-fold or more that of the fusion protein on the inner lumen surface of other EVs (e.g., exosomes) similarly modified with conventional EV (e.g., exosome) proteins.

[0413] In some embodiments, the density of the fusion protein comprising the scaffold protein of the present disclosure, such as exosome lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more that of the fusion protein on EVs (e.g., exosomes) similarly modified with CD9.

[0414] In some embodiments, the density of the fusion protein comprising the scaffold protein of the present disclosure, such as exosome lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more that of the fusion protein on EVs (e.g., exosomes) similarly modified with CD63.

[0415] In some embodiments, the density of the fusion protein comprising the scaffold protein of the present disclosure, such as exosome lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more that of the fusion protein on EVs (e.g., exosomes) similarly modified with CD81.

[0416] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with PDGFR.

[0417] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with a GPI-anchored protein.

[0418] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with lactadherin.

[0419] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with LAMP2.

[0420] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with LAMP2B.

[0421] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with a conventional protein.

[0422] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., MARCKS, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than 1,000-fold that on EVs (e.g., exosomes) similarly modified with variants of conventional EV (e.g., exosome) proteins.

[0423] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than 1,000-fold that on EVs (e.g., exosomes) similarly modified with CD9.

[0424] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or its modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than 1,000-fold that on EVs (e.g., exosomes) similarly modified with CD63.

[0425] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than 1,000-fold that on EVs (e.g., exosomes) similarly modified with CD81.

[0426] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than 1,000-fold that on EVs (e.g., exosomes) similarly modified with PDGFR.

[0427] In some embodiments, the density of the fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a GPI-anchored protein.

[0428] In some embodiments, the density of the fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a galectin.

[0429] In some embodiments, the density of the fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with LAMP2.

[0430] In some embodiments, the density of the fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with LAMP2B.

[0431] In some embodiments, the density of the fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a conventional protein.

[0432] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., MARCKSL1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with conventional EV (e.g., exosome) proteins.

[0433] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with CD9.

[0434] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with CD63.

[0435] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with CD81.

[0436] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with PDGFR.

[0437] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosome lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more than that on EVs (e.g., exosomes) similarly modified with GPI-anchored proteins.

[0438] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more on EVs (e.g., exosomes) similarly modified with lactadherin.

[0439] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more on EVs (e.g., exosomes) similarly modified with LAMP2.

[0440] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more on EVs (e.g., exosomes) similarly modified with LAMP2B.

[0441] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more on EVs (e.g., exosomes) similarly modified with a conventional protein.

[0442] In some embodiments, the density of the presence of a fusion protein comprising a scaffold protein of the present disclosure, such as an exosomal lumen protein (e.g., BASP1, its variants, fragments, variants of fragments, or modifications), is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold, or more on EVs (e.g., exosomes) similarly modified with a variant of a conventional EV (e.g., exosome) protein.

[0443] In some embodiments, the density of the presence of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence disclosed herein (e.g., in Table 1) without N-terminal M is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with CD9. In some embodiments, the EVs (e.g., exosomes) comprise a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the density of the presence of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with CD9.

[0444] In some embodiments, the density of the presence of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence disclosed herein (e.g., in Table 1) without N-terminal M is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with CD63. In some embodiments, the EVs (e.g., exosomes) comprise a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the density of the presence of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with CD63.

[0445] In some embodiments, the density of the presence of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence without N-terminal M disclosed herein (e.g., Table 1) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with CD81. In some embodiments, the EVs (e.g., exosomes) comprise a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the density of the presence of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with CD81.

[0446] In some embodiments, the density of the presence of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence without N-terminal M disclosed herein (e.g., Table 1) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with PDGFR. In some embodiments, the EVs (e.g., exosomes) comprise a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the density of the presence of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with PDGFR.

[0447] In some embodiments, the presence density of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence without N-terminal M disclosed herein (e.g., in Table 1) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a GPI-anchored protein. In some embodiments, the EVs (e.g., exosomes) comprise a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the presence density of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a GPI-anchored protein.

[0448] In some embodiments, the presence density of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence without N-terminal M disclosed herein (e.g., in Table 1) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a galactoside-binding lectin. In some embodiments, the EVs (e.g., exosomes) comprise a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the presence density of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a galactoside-binding lectin.

[0449] In some embodiments, the density of the presence of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence without N-terminal M disclosed herein (e.g., in Table 1) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with LAMP2. In some embodiments, the EVs (e.g., exosomes) comprise a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the density of the presence of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with LAMP2.

[0450] In some embodiments, the density of the presence of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence without N-terminal M disclosed herein (e.g., in Table 1) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with LAMP2B. In some embodiments, the EVs (e.g., exosomes) comprise a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the density of the presence of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with LAMP2Bt.

[0451] In some embodiments, the density of the presence of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence without N-terminal M disclosed herein (e.g., in Table 1) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a conventional protein. In some embodiments, the EV (e.g., exosome) comprises a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the density of the presence of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a conventional protein.

[0452] In some embodiments, the density of the presence of a fusion protein comprising any one of SEQ ID NOs: 1-109, the corresponding sequence without N-terminal M, or any scaffold protein sequence without N-terminal M disclosed herein (e.g., in Table 1) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a conventional EV protein (e.g., exosome protein). In some embodiments, the EV (e.g., exosome) comprises a fusion protein comprising any one of SEQ ID NOs: 1-109, wherein the fusion protein does not comprise an N-terminal Met, and wherein the density of the presence of the fusion protein is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more on EVs (e.g., exosomes) similarly modified with a conventional EV protein (e.g., exosome protein).

[0453] In some embodiments, the lumen-engineered EVs (e.g., exosomes) described herein exhibit superior properties compared to lumen-engineered EVs (e.g., exosomes) known in the art. For example, lumen-engineered EVs (e.g., exosomes) generated using the newly identified EV (e.g., exosome) proteins provided herein comprise modified proteins that are more highly enriched on their lumen surfaces compared to EVs (e.g., exosomes) at the prior art level (e.g., those generated using conventional EV (e.g., exosome) proteins).

[0454] In addition, compared to lumen-engineered EVs (e.g., exosomes) known in the art, the lumen-engineered EVs (e.g., exosomes) of the present disclosure may have greater, more specific, or more controlled biological activity. For example, lumen-engineered EVs (e.g., exosomes) containing a payload (e.g., a therapeutic or biologically relevant exogenous sequence fused to an EV (e.g., exosome) protein or a fragment thereof (e.g., a scaffold of the present disclosure such as BASP1 or a fragment, variant, or derivative thereof)) may have more desirable engineered features than fusions with scaffolds known in the art.

[0455] Scaffold proteins known in the art include tetraspanin molecules (e.g., CD63, CD81, CD9, etc.), lysosome-associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI-anchored proteins, galectins, and fragments thereof, as well as peptides having an affinity for any of these proteins or fragments thereof. For the avoidance of doubt, PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporters, or fragments or variants thereof are not conventional EV (e.g., exosome) proteins. Previously, overexpression of exogenous proteins relied on randomly or arbitrarily disposing the exogenous proteins into exosomes to generate lumen-engineered exosomes. This led to low levels and unpredictable densities of exogenous proteins in exosomes. Thus, the EV (e.g., exosome) proteins and fragments thereof described herein provide important advances in novel EV (e.g., exosome) compositions and methods for their preparation.

[0456] The fusion proteins provided herein may comprise a scaffold protein of the present disclosure, such as an exosome lumen protein, such as MARCKS, MARCKSL1, BASP1, or a fragment, variant, or derivative thereof, and an additional peptide. The additional peptide may be attached to the N-terminus or C-terminus of the EV (e.g., exosome) protein (scaffold protein) or a fragment, variant, or derivative thereof.

[0457] In some embodiments, the fusion proteins provided herein may comprise a scaffold protein of the present disclosure, such as MARCKS, MARCKSL1, BASP1, or a fragment, variant, or derivative thereof, and two additional peptides. Both additional peptides may be attached to the N-terminus or C-terminus of the EV (e.g., exosome) protein (scaffold protein) or a fragment, variant, or derivative thereof. In some embodiments, one of the two additional peptides is attached to the N-terminus and the other of the two additional peptides is attached to the C-terminus of the EV (e.g., exosome) protein (scaffold protein) or a fragment, variant, or derivative thereof.

[0458] In some embodiments, the compositions and methods for generating lumen-engineered extracellular vesicles described herein include nanovesicles.

[0459] IV. Producer cells for generating lumen-engineered EVs (e.g., exosomes)

[0460] The EVs (e.g., exosomes) of the present disclosure can be generated from in vitro grown cells or body fluids of a subject. When generating EVs (e.g., exosomes) from in vitro cell cultures, various producer cells such as HEK293, the EVs can be used in the present disclosure. Other cell types that can be used to generate the lumen-engineered EVs (e.g., exosomes) described herein include, but are not limited to, mesenchymal stem cells, T cells, B cells, dendritic cells, macrophages, and cancer cell lines.

[0461] Accordingly, the present disclosure provides cells for generating the EVs (e.g., exosomes) of the present disclosure. In some embodiments, the cells comprise one or more vectors, wherein the vector comprises a nucleic acid sequence encoding a scaffold protein and a payload (e.g., a bioactive molecule). In some embodiments, a nucleic acid sequence encodes a scaffold protein and a second nucleic acid sequence encodes a payload, e.g., a bioactive molecule. In some embodiments, the nucleic acid sequence encoding the scaffold protein and the nucleic acid sequence encoding the payload (e.g., a bioactive molecule) are in a single open reading frame; thus, the expression product will be a fusion protein comprising the scaffold protein fused to the payload (e.g., a bioactive molecule). In some embodiments, the nucleic acid sequence is operably linked to a promoter.

[0462] Producer cells can be genetically modified to contain one or more exogenous sequences to generate lumen-engineered EVs (e.g., exosomes). The genetically modified producer cells can be transiently or stably transformed to contain the exogenous sequence. The exogenous sequence can be transformed as a plasmid. The exogenous sequence can be stably integrated into a target site or a random site in the genomic sequence of the producer cell. In some embodiments, stable cell lines are generated for generating lumen-engineered EVs (e.g., exosomes).

[0463] The exogenous sequence can be inserted into the genomic sequence of the producer cell within the upstream (5'-end) or downstream (3'-end) of the endogenous sequence encoding the EV (e.g., exosome) protein. Various methods known in the art can be used to introduce the exogenous sequence into the producer cell. For example, cells modified using various gene editing methods (e.g., methods using homologous recombination, transposon-mediated systems, loxP-Cre systems, CRISPR / Cas9, or TALENs) are within the scope of the present disclosure.

[0464] The exogenous sequence may include a sequence encoding a scaffold protein of the present disclosure (e.g., an EV (e.g., exosome) protein or a modification or fragment of an EV (e.g., exosome) protein). An additional copy of the sequence encoding the scaffold protein (e.g., an EV (e.g., exosome) protein) may be introduced to produce lumen-engineered EVs (e.g., exosomes) having a higher density of EV (e.g., exosome) proteins. An exogenous sequence encoding a modification or fragment of an EV (e.g., exosome) protein may be introduced to produce lumen-engineered EVs (e.g., exosomes) comprising a modification or fragment of an EV (e.g., exosome) protein. An exogenous sequence encoding an affinity tag may be introduced to produce lumen-engineered EVs (e.g., exosomes) comprising a fusion protein that includes an affinity tag attached to an EV (e.g., exosome) protein.

[0465] In some embodiments, the lumen-engineered EVs (e.g., exosomes) have a higher density of EV (e.g., exosome) proteins (scaffold proteins) compared to native EVs (e.g., exosomes) isolated from the same or a similar producer cell type. In some embodiments, the density of the EV (e.g., exosome) protein (scaffold protein) present on the lumen-engineered EVs (e.g., exosomes) is 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 200-fold, 400-fold, 800-fold, 1,000-fold or more than that of native EVs <(e.g., exosomes). In some embodiments, the density of the EV (e.g., exosome) protein (scaffold protein) present on the lumen-engineered EVs (e.g., exosomes) is 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or more than that of native EVs (e.g., exosomes). In some embodiments, the density of the fusion protein comprising an EV (e.g., exosome) protein (scaffold protein) present on the lumen-engineered EVs (e.g., exosomes) is 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or more than that of the unmodified EV (e.g., exosome) protein (scaffold protein) on native EVs (e.g., exosomes). In some embodiments, the density of a fragment or variant of an EV (e.g., exosome) protein (scaffold protein) present on the lumen-engineered EVs (e.g., exosomes) is 2 to 4-fold, 4 to 8-fold, 8 to 16-fold, 16 to 32-fold, 32 to 64-fold, 64 to 100-fold, 100 to 200-fold, 200 to 400-fold, 400 to 800-fold, 800 to 1,000-fold or more than that of the unmodified EV (e.g., exosome) protein (scaffold protein) on native EVs (e.g., exosomes).

[0466] In some embodiments, the density of the presence of MARCKS, a fragment or variant thereof, or a modification thereof on the lumen-engineered EVs (e.g., exosomes) is 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times, or more than 1,000 times that of unmodified MARCKS on the native EVs (e.g., exosomes). In some embodiments, the density of the presence of MARCKSL1, a fragment or variant thereof, or a modification thereof on the lumen-engineered EVs (e.g., exosomes) is 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times, or more than 1,000 times that of unmodified MARCKSL1 on the native EVs (e.g., exosomes). In some embodiments, the density of the presence of BASP1, a fragment or variant thereof, or a modification thereof on the lumen-engineered EVs (e.g., exosomes) is 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times, or more than 1,000 times that of unmodified BASP1 on the native EVs (e.g., exosomes).

[0467] In some embodiments, the producer cells are further modified to contain additional exogenous sequences. For example, additional exogenous sequences can be included to regulate endogenous gene expression, or to produce exosomes that contain a certain polypeptide as a payload. In some embodiments, the producer cells are modified to contain two exogenous sequences, one encoding an EV (e.g., exosome) protein (scaffold protein) or a modification or fragment of an EV (e.g., exosome) protein (scaffold protein), and the other encoding a payload.

[0468] More specifically, lumen-engineered EVs (e.g., exosomes) can be produced from cells transformed with a sequence encoding one or more exosome lumen proteins (scaffold proteins), which include but are not limited to: (1) myristoylated alanine-rich protein kinase C substrate (MARCKS); (2) myristoylated alanine-rich protein kinase C substrate-like protein 1 (MARCKSL1); and (3) brain acid-soluble protein 1 (BASP1). Any of the one or more EV (e.g., exosome) lumen proteins (scaffold proteins) described herein can be expressed from a plasmid, an exogenous sequence inserted into the genome, or other exogenous nucleic acids such as synthetic messenger RNA (mRNA).

[0469] In some embodiments, the one or more EV (e.g., exosome) lumen proteins (scaffold proteins) are expressed in cells transformed with an exogenous sequence encoding their full-length endogenous form. In some embodiments, such an exogenous sequence encodes the MARCKS protein of SEQ ID NO:1 or a corresponding sequence lacking the N-terminal M. In certain aspects, such an exogenous sequence encodes the MARCKS protein of SEQ ID NO:1. In some embodiments, such an exogenous sequence encodes the MARCKS protein of SEQ ID NO:1, wherein the MARKS protein encoded by the exogenous sequence does not contain an N-terminal Met. In some embodiments, such an exogenous sequence encodes the MARCKSL1 protein of SEQ ID NO:2 or a corresponding sequence lacking the N-terminal M. In certain aspects, such an exogenous sequence encodes the MARCKSL1 protein of SEQ ID NO:2. In some embodiments, such an exogenous sequence encodes the MARCKSL1 protein of SEQ ID NO:2, wherein the MARCKSL1 protein encoded by the exogenous sequence does not contain an N-terminal Met. In some embodiments, such an exogenous sequence encodes the BASP1 protein of SEQ ID NO:3 or a corresponding sequence lacking the N-terminal M. In certain aspects, such an exogenous sequence encodes the BASP1 protein of SEQ ID NO:3. In some embodiments, such an exogenous sequence encodes the BASP1 protein of SEQ ID NO:3, wherein the BASP1 protein encoded by the exogenous sequence does not contain an N-terminal Met.

[0470] Lumen-engineered EV (e.g., exosomes) can be produced from cells transformed with a polynucleotide sequence encoding a fragment of one or more EV (e.g., exosome) lumen proteins (scaffold proteins), the exosome lumen proteins including but not limited to (1) myristoylated alanine-rich protein kinase C substrate (MARCKS); (2) myristoylated alanine-rich protein kinase C substrate-like protein 1 (MARCKSL1); and (3) brain acid-soluble protein 1 (BASP1).

[0471] In some embodiments, the polynucleotide sequence encodes a fragment of an EV (e.g., exosome) luminal protein (e.g., a scaffolding protein such as MARCKS, MARCKSL1, or BASP1) that is missing at least 5, 10, 50, 100, 200, or 300 amino acids from the N-terminus of the native protein. In some embodiments, the polynucleotide sequence encodes a fragment of an EV (e.g., exosome) luminal protein (e.g., a scaffolding protein such as MARCKS, MARCKSL1, or BASP1) that is missing at least 5, 10, 50, 100, 200, or 300 amino acids from the C-terminus of the native protein. In some embodiments, the polynucleotide sequence encodes a fragment of an EV (e.g., exosome) luminal protein (e.g., a scaffolding protein such as MARCKS, MARCKSL1, or BASP1) that is missing at least 5, 10, 50, 100, 200, or 300 amino acids from both the N-terminus and the C-terminus of the native protein. In some embodiments, the polynucleotide sequence encodes a fragment of an EV (e.g., exosome) luminal protein (e.g., a scaffolding protein such as MARCKS, MARCKSL1, or BASP1) that is missing one or more functional or structural domains of the native protein.

[0472] In some embodiments, the fusion protein comprises a peptide of SEQ ID NO: 4-109, the corresponding sequence without an N-terminal M, or any scaffold protein sequence without an N-terminal M disclosed herein (e.g., in Table 1). In some embodiments, the fusion protein comprises a peptide of SEQ ID NO: 4-109 or any scaffold protein sequence disclosed herein (e.g., in Table 1), wherein the fusion protein does not comprise an N-terminal Met. In some embodiments, the fusion protein comprises a peptide of SEQ ID NO: 13. In some embodiments, the fusion protein comprises a peptide of SEQ ID NO: 13, wherein the fusion protein does not comprise an N-terminal Met. In some embodiments, the fusion protein comprises a scaffold protein, such as an exosome lumen protein, which comprises a peptide having the sequence MGXKLSKKK (SEQ ID NO: 117) or GXKLSKKK (SEQ ID NO: 425), where X is alanine or any other amino acid. In some embodiments, the fusion protein comprises a scaffold protein, such as an exosome lumen protein, which comprises a peptide having the sequence GXKLSKKK (SEQ ID NO: 425), where X is alanine or any other amino acid, and wherein the scaffold protein, such as an exosome lumen protein, does not comprise an N-terminal Met. In some embodiments, the fusion protein comprises a scaffold protein, such as an exosome lumen protein, which comprises a peptide having the sequence GXKLSKKK (SEQ ID NO: 425), where X is alanine or any other amino acid, and wherein the fusion protein does not comprise an N-terminal Met. In some embodiments, the fusion protein comprises a scaffold protein that comprises the amino acid sequence GXKLSKKK (SEQ ID NO: 425), where X is alanine or any other amino acid, and wherein the scaffold protein does not have a Met at the N-terminus of the amino acid sequence, e.g., comprises MGXKLSKKK (SEQ ID NO: 117). In other embodiments, the fusion protein comprises a scaffold protein, such as an exosome lumen protein, which comprises the amino acid sequence GXKLSKKK (SEQ ID NO: 425), wherein the fusion protein does not comprise MGXKLSKKK.In some embodiments, the fusion protein comprises a scaffold protein, the scaffold protein comprising a peptide having the sequence (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+) or (G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), where each position within the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu). In some embodiments, the fusion protein comprises a scaffold protein, such as an exosome lumen protein, having the amino acid sequence (G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), where the fusion protein does not comprise (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+). In some embodiments, the fusion protein comprises a scaffold protein, such as an exosome lumen protein, which comprises a peptide having the sequence (M)(G)(π)(X)(Φ / π)(π)(+)(+) or (G)(π)(X)(Φ / π)(π)(+)(+), where each position within the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu). In other embodiments, the fusion protein comprises a scaffold protein, such as an exosome lumen protein, which comprises the amino acid sequence (G)(π)(X)(Φ / π)(π)(+)(+), where the fusion protein does not comprise (M)(G)(π)(X)(Φ / π)(π)(+)(+).

[0473] In some embodiments, lumen-engineered EVs (e.g., exosomes) can be produced from cells transformed with the following sequences that encode an EV (e.g., exosome) protein (e.g., a scaffolding protein such as an exosome lumen protein, such as MARCKS, MARCKSL1, or BASP1) or a fragment or modification thereof fused to one or more heterologous proteins. In some embodiments, the one or more heterologous proteins are fused to the N-terminus of an EV (e.g., exosome) protein (e.g., a scaffolding protein such as MARCKS, MARCKSL1, or BASP1) or a modification thereof, particularly a fragment or variant thereof. In some embodiments, the one or more heterologous proteins are fused to the C-terminus of an EV (e.g., exosome) protein (e.g., a scaffolding protein such as MARCKS, MARCKSL1, or BASP1) or a modification thereof, particularly a fragment or variant thereof. In some embodiments, the one or more heterologous proteins are fused to both the N-terminus and the C-terminus of an EV (e.g., exosome) protein (e.g., a scaffolding protein such as MARCKS, MARCKSL1, or BASP1) or a modification thereof, particularly a fragment or variant thereof. In some embodiments, the one or more heterologous proteins are mammalian proteins. In some embodiments, the one or more heterologous proteins are human proteins.

[0474] In some embodiments, lumen-engineered EVs (e.g., exosomes) are produced from cells transformed with a sequence encoding a polypeptide having a sequence identical or similar to the full-length or a fragment of a native EV (e.g., exosome) lumen protein (scaffolding protein), the lumen protein including but not limited to,

[0475] (1) Myristoylated alanine-rich C kinase substrate (MARCKS);

[0476] (2) Myristoylated alanine-rich C kinase substrate-like protein 1 (MARCKSL1); and

[0477] (3) Brain acid-soluble protein 1 (BASP1).

[0478] In some embodiments, the polypeptide has at least about 50% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has 50% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M. In some embodiments, the polypeptide has at least about 55% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has at least about 55% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M. In some embodiments, the polypeptide has at least about 60% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has at least about 60% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M. In some embodiments, the polypeptide has at least about 65% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has at least about 65% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M. In some embodiments, the polypeptide has at least about 70% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has at least about 70% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M. In some embodiments, the polypeptide has at least about 75% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has at least about 75% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M. In some embodiments, the polypeptide has at least about 80% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has at least about 80% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M. In some embodiments, the polypeptide has at least about 85% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has at least about 85% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M. In some embodiments, the polypeptide has at least about 90% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has at least about 90% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M. In some embodiments, the polypeptide has at least about 95% identity with the full length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., has at least about 95% identity with SEQ ID NO: 1-3 or any corresponding sequence without the N-terminal M.In some embodiments, the polypeptide has at least about 99% identity to the full-length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., at least about 99% identity to SEQ ID NO: 1-3 or any corresponding sequence lacking the N-terminal M. In some embodiments, the polypeptide has at least about 99.9% identity to the full-length or a fragment of a native EV (e.g., exosome) luminal protein (scaffold protein), e.g., at least about 99.9% identity to SEQ ID NO: 1-3 or any corresponding sequence lacking the N-terminal M.

[0479] In some embodiments, lumen-engineered EVs (e.g., exosomes) produced from cells contain a polypeptide having a sequence identical or similar to a fragment of brain acid-soluble protein 1 (BASP1). In some embodiments, the polypeptide has at least about 50% identity to the full-length or a fragment of BASP1, e.g., has at least about 50% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 55% identity to the full-length or a fragment of BASP1, e.g., has at least about 50% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 60% identity to the full-length or a fragment of BASP1, e.g., has at least about 60% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 65% identity to the full-length or a fragment of BASP1, e.g., has at least about 65% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 70% identity to the full-length or a fragment of BASP1, e.g., has at least about 70% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 75% identity to the full-length or a fragment of BASP1, e.g., has at least about 75% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 80% identity to the full-length or a fragment of BASP1, e.g., has at least about 80% identity to SEQ ID NO4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 85% identity to the full-length or a fragment of BASP1, e.g., has at least about 85% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1).In some embodiments, the polypeptide has at least about 90% identity to the full length or a fragment of BASP1, e.g., has at least about 90% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 95% identity to the full length or a fragment of BASP1, e.g., has at least about 95% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 99% identity to the full length or a fragment of BASP1, e.g., has at least about 99% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has at least about 99.9% identity to the full length or a fragment of BASP1, e.g., has at least about 99.9% identity to SEQ ID NOs: 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1). In some embodiments, the polypeptide has about 100% identity to a fragment of BASP1, e.g., has about 100% identity to 4-109, the corresponding sequences without the N-terminal M, or any BASP1 sequence without the N-terminal M described herein (e.g., in Table 1).

[0480] In some embodiments, the lumen-engineered EVs (e.g., exosomes) produced from cells contain a polypeptide having a sequence identical or similar to a fragment of brain acid-soluble protein 1 (BASP1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 50% identity to the full-length or a fragment of BASP1, e.g., has at least about 50% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 55% identity to the full-length or a fragment of BASP1, e.g., has at least about 55% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 60% identity to the full-length or a fragment of BASP1, e.g., has at least about 60% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 65% identity to the full-length or a fragment of BASP1, e.g., has at least about 65% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 70% identity to the full-length or a fragment of BASP1, e.g., has at least about 70% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 75% identity to the full-length or a fragment of BASP1, e.g., has at least about 75% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 80% identity to the full-length or a fragment of BASP1, e.g., has at least about 80% identity to SEQ ID NO4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 85% identity to the full-length or a fragment of BASP1, e.g., has at least about 85% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met.In some embodiments, the polypeptide has at least about 90% identity to the full-length or a fragment of BASP1, e.g., has at least about 90% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 95% identity to the full-length or a fragment of BASP1, e.g., has at least about 95% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 99% identity to the full-length or a fragment of BASP1, e.g., has at least about 99% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has at least about 99.9% identity to the full-length or a fragment of BASP1, e.g., has at least about 99.9% identity to SEQ ID NO:4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met. In some embodiments, the polypeptide has about 100% identity to a fragment of BASP1, e.g., has about 100% identity to 4-109 or any BASP1 sequence described herein (e.g., in Table 1), wherein the polypeptide does not contain an N-terminal Met.

[0481] VI. Preparation Method

[0482] The EVs (e.g., exosomes) of the present disclosure can be produced by chemical synthesis, recombinant DNA technology, biochemical or enzymatic fragmentation of larger molecules, combinations of the above techniques, or by any other method. In one embodiment, the present disclosure provides a method for conjugating a bioactive molecule to an EV (e.g., an exosome). The method includes linking the bioactive molecule to the EV (e.g., an exosome) as described above.

[0483] In some embodiments of the present disclosure, the EVs (e.g., exosomes) of the present disclosure can be prepared using the producer cells described above. Accordingly, the present disclosure provides a method for preparing EVs (e.g., exosomes), which includes culturing the producer cells disclosed herein under suitable conditions and obtaining the EVs (e.g., exosomes) of the present disclosure.

[0484] In other embodiments, the scaffold proteins of the present disclosure, such as exosomal lumen proteins, can be recombinantly produced and subsequently incorporated into the EVs (e.g., exosomes) of the present disclosure. In other embodiments, only the polypeptide portion of the scaffold protein is recombinantly produced. In some embodiments, the polypeptide portion of the recombinantly produced scaffold protein is subsequently modified, e.g., chemically or enzymatically, to incorporate a membrane anchor (e.g., N-terminal myristic acid). In some embodiments, the semi-recombinant (i.e., the recombinant product of the combined polypeptide portion, followed by chemical or enzymatic modification) produced scaffold protein is subsequently incorporated into the EVs (e.g., exosomes) of the present disclosure.

[0485] In other embodiments, the scaffold proteins of the present disclosure, such as exosomal lumen proteins, can be chemically produced, e.g., using solid-phase peptide synthesis, and subsequently incorporated into the EVs (e.g., exosomes) of the present disclosure. In other embodiments, only the polypeptide portion of the scaffold protein is synthesized. In some embodiments, the polypeptide portion of the synthesized scaffold protein is subsequently modified, e.g., chemically or enzymatically, to incorporate a membrane anchor (e.g., N-terminal myristic acid). In some embodiments, the semi-synthetic (i.e., the synthetic product of the combined polypeptide portion, followed by chemical or enzymatic modification) produced scaffold protein is subsequently incorporated into the EVs (e.g., exosomes) of the present disclosure.

[0486] In other embodiments, the scaffold proteins of the present disclosure, such as exosomal lumen proteins, can be produced using cell-free expression, such as a reticulocyte system, in vitro and subsequently incorporated into the EVs (e.g., exosomes) of the present disclosure. In other embodiments, only the polypeptide portion of the scaffold protein is produced in vitro, e.g., in a cell-free system. In some embodiments, the polypeptide portion of the scaffold protein produced in vitro, e.g., in a cell-free system, is subsequently modified, e.g., chemically or enzymatically, to incorporate a membrane anchor (e.g., N-terminal myristic acid). In some embodiments, the semi-in vitro (i.e., the in vitro product of the combined polypeptide portion, followed by chemical or enzymatic modification) produced scaffold protein is subsequently incorporated into the EVs (e.g., exosomes) of the present disclosure.

[0487] In some embodiments, the methods described herein further include the step of characterizing the EVs (e.g., exosomes) contained in each collected fraction during their production and purification. In some embodiments, the contents of the EVs (e.g., exosomes) can be extracted for research and characterization. In some embodiments, the EVs (e.g., exosomes) are isolated and characterized by metrics including but not limited to size, shape, morphology, or molecular composition such as nucleic acids, proteins, metabolites, and lipids.

[0488] VII. Pharmaceutical Compositions and Methods of Administration

[0489] The present disclosure also provides a pharmaceutical composition suitable for administration to a subject, comprising the EVs (e.g., exosomes) described herein. The pharmaceutical composition generally comprises a plurality of EVs (e.g., exosomes) and a pharmaceutically acceptable excipient or carrier in a form suitable for administration to a subject, and the EVs comprise at least one payload covalently or non-covalently linked to the plurality of EVs (e.g., exosomes), such as, for example, a bioactive molecule.

[0490] The pharmaceutically acceptable excipient or carrier is determined in part by the particular composition being administered and the particular method for administering that composition. Accordingly, there are a variety of suitable formulations of pharmaceutical compositions comprising a plurality of EVs (e.g., exosomes). (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)). Pharmaceutical compositions are generally formulated to be sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. In some embodiments, the pharmaceutical composition comprises one or more chemical compounds, such as, for example, small molecules covalently linked to the EVs (e.g., exosomes) described herein.

[0491] In some embodiments, the pharmaceutical composition comprises one or more therapeutic or diagnostic agents and the EVs (e.g., exosomes) described herein. In certain embodiments, the EVs (e.g., exosomes) are co-administered with one or more additional therapeutic or diagnostic agents in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprising the EVs (e.g., exosomes) is administered before the additional therapeutic or diagnostic agent. In some embodiments, the pharmaceutical composition comprising the EVs (e.g., exosomes) is administered after the additional therapeutic or diagnostic agent. In further embodiments, the pharmaceutical composition comprising the EVs (e.g., exosomes) is co-administered with the additional therapeutic or diagnostic agent.

[0492] The present disclosure provides a pharmaceutical composition in a form suitable for administration to a subject, comprising the EVs (e.g., exosomes) of the present disclosure having a desired purity and a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable excipient or carrier can be determined in part by the particular composition being administered and the particular method for administering that composition. Accordingly, there are a variety of suitable formulations of pharmaceutical compositions comprising a plurality of extracellular vesicles. (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st ed. (2005)). Pharmaceutical compositions are generally formulated to be sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0493] In some embodiments, the pharmaceutical composition comprises one or more therapeutic or diagnostic agents and EVs (e.g., exosomes) as described herein. In certain embodiments, the EVs (e.g., exosomes) are co-administered with one or more additional therapeutic or diagnostic agents in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered before the additional therapeutic or diagnostic agent is administered. In some embodiments, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered after the additional therapeutic or diagnostic agent is administered. In additional embodiments, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered simultaneously with the additional therapeutic or diagnostic agent.

[0494] Acceptable carriers, excipients or stabilizers are non-toxic to the recipient (e.g., animal or human) at the dosages and concentrations employed and include buffering agents such as phosphates, citrates and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins, etc.; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0495] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Unless any conventional medium or compound is incompatible with the extracellular vesicles described herein, its use in the composition can be considered. Supplementary therapeutic agents can also be incorporated into the composition. Generally, the pharmaceutical composition is formulated to be compatible with its intended route of administration. The EVs (e.g., exosomes) of the present disclosure can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intratumoral, intramuscular routes or as an inhalant. In certain embodiments, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered intravenously, for example, by injection. The EVs (e.g., exosomes) can optionally be administered in combination with other therapeutic agents that are at least partially effective in treating the disease, disorder, or condition for which the EVs (e.g., exosomes) are targeted.

[0496] Solutions or suspensions may include the following components: a sterile diluent such as water, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial compound such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating compound such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and a compound for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. The preparation can be enclosed in an ampoule, a disposable syringe, or a multi-dose vial made of glass or plastic.

[0497] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition is generally sterile and fluid to the extent that it is readily injectable. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using coatings such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants, appropriate fluidity can be maintained. The action of microorganisms can be prevented by various antibacterial and antifungal compounds (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). If desired, isotonic compounds such as sugars, polyols such as mannitol, sorbitol, and sodium chloride can be added to the composition. Prolonged absorption of the injectable composition can be caused by including compounds that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0498] A sterile injectable solution can be prepared by incorporating the EVs (e.g., exosomes) of the present disclosure in an effective amount and in a suitable solvent into one or a combination of the ingredients listed herein as needed. Generally, a dispersion is prepared by incorporating the EVs (e.g., exosomes) into a sterile vehicle that contains a basic dispersion medium and any other desired ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any other desired ingredients (from its previously sterile filtered solution). The EVs (e.g., exosomes) can be administered in the form of depot injection or implant formulations, which can be formulated in a manner that allows for sustained release or pulsed release of the EVs (e.g., exosomes).

[0499] Systemic administration of a composition comprising the EVs (e.g., exosomes) of the present disclosure can also be by the transmucosal route. For transmucosal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished by using, for example, a nasal spray.

[0500] In certain embodiments, a pharmaceutical composition comprising the EVs (e.g., exosomes) of the present disclosure is administered intravenously to a subject who can benefit from the pharmaceutical composition. In certain other embodiments, the composition is administered to the lymphatic system, for example, by intralymphatic injection or by intranodal injection (see, e.g., Senti et al., PNAS 105(46):17908 (2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus or liver.

[0501] In certain embodiments, a pharmaceutical composition comprising the EVs (e.g., exosomes) of the present disclosure is administered in the form of a liquid suspension. In certain embodiments, the pharmaceutical composition is administered in the form of a formulation capable of forming a depot after administration. In certain preferred embodiments, the depot slowly releases the EVs (e.g., exosomes) into the circulation or remains in the form of a depot.

[0502] Generally, a pharmaceutically acceptable composition is highly purified to be free of contaminants, is biocompatible and non-toxic, and is suitable for administration to a subject. If water is an ingredient of the carrier, the water is highly purified and treated to be free of contaminants (e.g., endotoxins).

[0503] The pharmaceutically acceptable carrier can be lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and / or mineral oil, but is not limited thereto. The pharmaceutical composition may also contain lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, and / or preservatives.

[0504] The pharmaceutical compositions described herein comprise the EVs (e.g., exosomes) described herein and optionally a pharmaceutically active agent or therapeutic agent and / or diagnostic agent. The therapeutic agent can be a biological agent, a small molecule agent, or a nucleic acid agent. The diagnostic agent can be, for example, a contrast agent.

[0505] Dosage forms comprising the pharmaceutical compositions are provided, the pharmaceutical compositions comprising the EVs (e.g., exosomes) described herein. In some embodiments, the dosage form is formulated as a liquid suspension for intravenous injection. In some embodiments, the dosage form is formulated as a liquid suspension for intratumoral injection.

[0506] In certain embodiments, the EV (e.g., exosome) preparation of the present disclosure is subjected to irradiation, such as X-rays, γ-rays, β-particles, α-particles, neutrons, protons, elemental nuclei, ultraviolet light, to destroy residual replication-competent nucleic acids.

[0507] In certain embodiments, the EV (e.g., exosome) preparation of the present disclosure is γ-irradiated with an irradiation dose of more than 1 kGy, 5 kGy, 10 kGy, 15 kGy, 20 kGy, 25 kGy, 30 kGy, 35 kGy, 40 kGy, 50 kGy, 60 kGy, 70 kGy, 80 kGy, 90 kGy, 100 kGy, or more than 100 kGy.

[0508] In certain embodiments, the EV (e.g., exosome) preparations of the present disclosure are irradiated with X-rays using an irradiation dose of more than 0.1 kGy, 0.5 kGy, 1 kGy, 5 kGy, 10 kGy, 15 kGy, 20 kGy, 25 kGy, 30 kGy, 35 kGy, 40 kGy, 50 kGy, 60 kGy, 70 kGy, 80 kGy, 90 kGy, 100 kGy, 200 kGy, 300 kGy, 400 kGy, 500 kGy, 600 kGy, 700 kGy, 800 kGy, 900 kGy, 1000 kGy, 2000 kGy, 3000 kGy, 4000 kGy, 5000 kGy, 6000 kGy, 7000 kGy, 8000 kGy, 9000 kGy, 10000 kGy or more.

[0509] The EVs (e.g., exosomes) of the present disclosure can be used in combination with other drugs. Specifically, the EVs (e.g., exosomes) of the present disclosure can be used in combination with drugs such as hormonal therapeutic agents, chemotherapeutic agents, immunotherapeutic agents, drugs that inhibit the action of cell growth factors or cell growth factor receptors, and the like.

[0510] VIII. Therapeutic Uses

[0511] The present disclosure provides methods for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising the EVs (e.g., exosomes) of the present disclosure. The present disclosure also provides methods for preventing or ameliorating the symptoms of a disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising the EVs (e.g., exosomes) of the present disclosure. The present disclosure further provides methods for diagnosing a disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising the EVs (e.g., exosomes) of the present disclosure. Also provided are the EVs (e.g., exosomes) of the present disclosure for use in therapy, as a medicament, for treating a disease or disorder in a subject in need thereof, for preventing or ameliorating the symptoms of a disease or disorder in a subject in need thereof, or for diagnosing a disease or disorder in a subject in need thereof.

[0512] In one embodiment, the disease or disorder is cancer, an inflammatory disease, a neurodegenerative disorder, a central nervous disease, or a metabolic disease.

[0513] The present disclosure also provides methods of preventing and / or treating a disease or disorder in a subject in need thereof, which comprise administering to the subject an EV (e.g., exosome) disclosed herein. In some embodiments, the diseases or disorders treatable by the methods of the invention include cancer, graft-versus-host disease (GvHD), autoimmune diseases, infectious diseases or fibrotic diseases. In some embodiments, the treatment is prophylactic. In other embodiments, the EVs (e.g., exosomes) of the present disclosure are used to induce an immune response. In other embodiments, the EVs (e.g., exosomes) of the present disclosure are used to vaccinate a subject. In some embodiments, the disease or disorder is cancer.

[0514] In some embodiments, the disease or disorder is an infectious disease. In certain embodiments, the disease or disorder is an oncovirus. In some embodiments, the infectious diseases treatable by the present disclosure include, but are not limited to, human gammaherpesvirus 4 (Epstein Barr virus), influenza A virus, influenza B virus, cytomegalovirus, Staphylococcus aureus, Mycobacterium tuberculosis, Chlamydia trachomatis, HIV-1, HIV-2, coronaviruses (e.g., MERS-CoV and SARS CoV), filoviruses (e.g., Marburg and Ebola viruses), Streptococcus pyogenes, Streptococcus pneumoniae, species of Plasmodium (e.g., Plasmodium vivax and Plasmodium falciparum), chikungunya virus, human papillomavirus (HPV), hepatitis B virus, hepatitis C virus, human herpesvirus 8, herpes simplex virus 2 (HSV2), a species of Klebsiella, Pseudomonas aeruginosa, a species of Enterococcus, a species of Proteus, a species of Enterobacter, a species of Actinobacter, coagulase-negative staphylococci (CoNS), a species of Mycoplasma, or a combination thereof.

[0515] In some embodiments, the EVs (e.g., exosomes) are administered intravenously into the circulatory system of the subject. In some embodiments, the EVs (e.g., exosomes) are infused and administered into the vein of the subject in a suitable liquid.

[0516] In some embodiments, EVs (e.g., exosomes) are administered intra-arterially to the circulatory system of a subject. In some embodiments, EVs (e.g., exosomes) are infused in a suitable liquid and administered into an artery of a subject.

[0517] In some embodiments, EVs (e.g., exosomes) are administered to a subject by intrathecal administration. In some embodiments, EVs (e.g., exosomes) are administered by injection into the spinal canal or subarachnoid space such that they reach the cerebrospinal fluid (CSF).

[0518] In some embodiments, EVs (e.g., exosomes) are administered intratumorally to one or more tumors of a subject.

[0519] In some embodiments, EVs (e.g., exosomes) are administered to a subject by intranasal administration. In some embodiments, EVs (e.g., exosomes) can be insufflated through the nose in a form of topical or systemic administration. In certain embodiments, EVs (e.g., exosomes) are administered in the form of a nasal spray.

[0520] In some embodiments, EVs (e.g., exosomes) are administered to a subject by intraperitoneal administration. In some embodiments, EVs (e.g., exosomes) are injected into a suitable liquid and injected into the peritoneum of a subject. In some embodiments, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) into lymphatic vessels. In some embodiments, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) into the thymus, spleen, and / or bone marrow. In some embodiments, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) into one or more lymph nodes. In some embodiments, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) into one or more of the cervical lymph nodes, inguinal lymph nodes, mediastinal lymph nodes, or sternal lymph nodes. In some embodiments, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) into the pancreas.

[0521] In some embodiments, EVs (e.g., exosomes) are administered to a subject by periocular administration. In some embodiments, EVs (e.g., exosomes) are injected into the periocular tissue. Periocular drug administration includes subconjunctival, anterior sub-Tenon’s, posterior sub-Tenon’s, and retrobulbar administration routes.

[0522] IX. Kit

[0523] The present disclosure also provides a kit or article comprising one or more EVs (e.g., exosomes) of the present disclosure and optionally instructions for use. In some embodiments, the kit or article comprises a pharmaceutical composition described herein, which comprises at least one EV (e.g., exosome) of the present disclosure and instructions for use.

[0524] In some embodiments, the kit or article comprises at least one EV (e.g., exosome) of the present disclosure or a pharmaceutical composition containing the EV (e.g., exosome) in one or more containers. Those skilled in the art will readily recognize that the EVs (e.g., exosomes) of the present disclosure, pharmaceutical compositions containing the EVs (e.g., exosomes) of the present disclosure, or combinations thereof can be readily incorporated into one of the established kit formats, which are well known in the art.

[0525] In some embodiments, the kit or article comprises (i) an EV (e.g., exosome), (ii) one or more payloads, such as bioactive molecules, (ii) a reagent that covalently attaches the one or more payloads (e.g., bioactive molecules) to the EV (e.g., exosome), or (iv) any combination thereof, and instructions for performing a reaction to covalently attach the one or more payloads (e.g., bioactive molecules) to the EV (e.g., exosome).

[0526] In some embodiments, the kit comprises a reagent for conjugating a payload (e.g., a bioactive molecule) to an EV (e.g., an exosome), and instructions for performing the conjugation.

[0527] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology within the skill of the art. Such techniques are explained in detail in the literature. See, for example, Sambrook et al., eds. (1989) Molecular Cloning A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press); Sambrook et al., eds. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover, ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); edited by Miller and Calos (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); edited by Wu et al., Methods In Enzymology, Volumes 154 and 155; edited by Mayer and Walker (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); edited by Weir and Blackwell, (1986) Handbook Of Experimental Immunology, Volumes I - IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986);); Crooke, Antisense drug Technology: Principles, Strategies and Applications, 2nd Edition CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0528] sequence

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540] Examples

[0541] The following examples are presented in order to provide a complete disclosure and description of how to make and use the present disclosure to those of ordinary skill in the art, and are not intended to limit the scope of what the inventors regard as the present disclosure, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should also be considered.

[0542] Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; nt, nucleotide, etc.

[0543] Unless otherwise indicated, the practice of the present disclosure will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the capabilities of those of ordinary skill in the art. Such techniques are explained in detail in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 21st ed. (Easton, Pennsylvania: Mack Publishing Company, 2005); Carey and Sundberg Advanced Organic Chemistry 3rd ed. (Plenum Press) Volumes A and B (1992).

[0544] Example 1

[0545] Identify the minimum protein sequence sufficient to load the lumenal exosome payload

[0546] To identify the minimal BASP1 amino acid sequence between the 12 - amino acid truncation that promotes loading and the 6 - amino acid truncation that fails to promote loading (as shown above), single truncation mutants of the N - terminus of BASP1 fused to tags and GFP were generated and stably expressed in HEK293SF cells ( Figure 1A ). As described above, exosomes were purified from the stable cell cultures. BASP1 sequences of seven to twelve amino acids were able to load GFP into exosomes at high density, while the first six amino acids were not ( Figure 1B ). These data indicate that at least one lysine residue after position six is required for luminal loading of exosomes with the N - terminus of BASP1 (i.e., attachment to the luminal surface of exosomes).

[0547] The serine at position 6 of BASP1 is highly conserved among species and in MARCKS and MARCKSL1. To determine whether this amino acid is required for loading payloads into exosomes, HEK293SF cells were stably transfected with expression plasmids encoding BASP1 1 - 30 - -GFP or BASP1 1 - 30 - -GFP, where BASP1 1 - 30 - -GFP or BASP1 1 - 30 - -GFP included point mutants (replacing serine with aspartic acid (S6D; polar charge substitution) or alanine (S6A; small non - polar substitution)). Additionally, the lysine at position 5 was mutated to glutamate (L5Q) to test the potential role of this position in regulating myristoylation, palmitoylation, and other membrane functions of several membrane - associated proteins (Gottlieb - Abraham et al., Mol. Biol. Cell. December 1, 2016; 27(24):3926 - 3936) ( Figure 2A ). BASP1 S6D completely abolished the loading of GFP into exosomes, while S6A did not alter the loading. BASP1 L5Q also did not affect luminal loading, indicating that the negative charge at position 6 disrupts loading, while polar amino acid substitutions at position 5 are well - tolerated ( Figure 2B ).

[0548] The first 30 amino acids of BASP1 contain the N - terminal leader sequence identified above, followed by a lysine - rich amino acid segment. To understand whether the N - termini of MARCKS and MARCKSL1 can load exosomes like BASP1, HEK293SF cells were transfected with full - length MARCKS and MARCKSL1 proteins or with The amino acids 1 - 30 fused with GFP were stably transfected into HEK293SF cells. The purified exosomes were analyzed by SDS PAGE and staining to determine the loading extent. Full-length MARCKS and MARCKSL1 were able to load exosomes with GFP, but amino acids 1 - 30 were worse than the full-length proteins, indicating that there are other structural or sequence features in the distal regions of MARCKS and MARCKSL1 proteins required for loading ( Figure 3 ). Sequence analysis of MARCKS and MARCKSL1 revealed regions with potential sequence homology to the N-terminus of BASP1.

[0549] Amino acids 152 - 173 of MARCKS and amino acids 87 - 110 of MARCKSL1 are lysine-rich and interspersed with phenylalanine and serine residues, and are expected to be phosphorylation site domains (PSD) or effector domains (ED) ( Figure 4 ). HEK293SF cells were stably transfected with a plasmid construct (MG-PSD) that fuses amino acids 1 - 3 of MARCKS with the PSD domain. Single point mutations were generated at the predicted myristoylation site (MA-PSD) and position 6 (K6S and K6A) to determine the role of these residues in exosome loading ( Figure 5A ). Western blotting of purified exosomes showed that neither MG-PSD nor MA-PSD could efficiently load exosomes compared to the positive control of BASP1 1 - 30. Interestingly, the K6A and K6S mutations led to improved loading, indicating that the positive charge at position 6 prevents the loading of exosome payloads, and the PSD of MARCKS can functionally complement the endogenous N-terminal sequence ( Figure 5B ). Collectively, these studies allowed the identification of several motifs sufficient to load payloads into exosomes ( Figure 6 ).

[0550] The narrowest motif, Motif 1, allows protein sequences without the first Met of (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118) or (G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:202), where each letter or group of letters inside the parentheses is an amino acid position, and where additionally position 5 cannot be a positively charged amino acid (K / R / H), and position 6 cannot be a negatively charged amino acid (D / E).

[0551] Sub-motifs of Motif 1 include, but are not limited to, the following protein sequences:

[0552] (M)(G)(G)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 180),

[0553] (G)(G)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 191)

[0554] (M)(G)(A)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 181),

[0555] (G)(A)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 192),

[0556] (M)(G)(S)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 182),

[0557] (G)(S)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 193),

[0558] (M)(G)(G / A / S)(K)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 183),

[0559] (G)(G / A / S)(K)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 194),

[0560] (M)(G)(G / A / S)(Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 184),

[0561] (G)(G / A / S)(Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO: 195),

[0562] (M)(G)(G / A / S)(K / Q)(L)(S / A)(K)(K) (SEQ ID NO: 185),

[0563] (G)(G / A / S)(K / Q)(L)(S / A)(K)(K) (SEQ ID NO: 196),

[0564] (M)(G)(G / A / S)(K / Q)(F)(S / A)(K)(K) (SEQ ID NO: 186),

[0565] (G)(G / A / S)(K / Q)(F)(S / A)(K)(K) (SEQ ID NO: 197),

[0566] (M)(G)(G / A / S)(K / Q)(S)(S / A)(K)(K) (SEQ ID NO: 187),

[0567] (G)(G / A / S)(K / Q)(S)(S / A)(K)(K) (SEQ ID NO: 198),

[0568] (M)(G)(G / A / S)(K / Q)(Q)(S / A)(K)(K) (SEQ ID NO: 188),

[0569] (G)(G / A / S)(K / Q)(Q)(S / A)(K)(K) (SEQ ID NO: 199),

[0570] (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S)(K)(K) (SEQ ID NO: 189),

[0571] (G)(G / A / S)(K / Q)(L / F / S / Q)(S)(K)(K) (SEQ ID NO: 200),

[0572] (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(A)(K)(K) (SEQ ID NO: 190) and

[0573] (G)(G / A / S)(K / Q)(L / F / S / Q)(A)(K)(K) (SEQ ID NO: 201),

[0574] where position 5 cannot be a positively charged amino acid (K / R / H), and position 6 cannot be a negatively charged amino acid (D / E).

[0575] Motif 2 (a more extensive motif) can be represented as (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+) or (G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+) without the first Met, where each position inside the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, GlV, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu). For the naming of amino acids, see R. Aasland et al., FEBS Letters 513 (2002): 141-144.

[0576] Motif 3 (the most extensive motif) can be represented as (M)(G)(π)(X)(Φ / π)(π)(+)(+) or (G)(π)(X)(Φ / π)(π)(+)(+) without the first Met, where each position inside the parentheses represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu). In all cases of Motifs 1-3, the sequence can be truncated by one amino acid to a length of seven total amino acids (i.e., consisting of amino acids 1-7 in the order presented in Motifs 1-3). Any sequence derived from any one of Motifs 1, 2, or 3 (or these motifs lacking amino acid 7) can be used to load the payload into exosomes to the same or comparable extent as full-length BASP1 or the native truncated sequences of BASP1. In-depth analysis of the amino acid sequence-structure-function provides novel insights into the need for producer cells to import bio-expressed payloads into exosomes.

[0577] Example 2

[0578] The N-terminus of BASP1 is sufficient to load different classes of proteins

[0579] The results in Example 1 indicate that the N-terminus of BASP1 can be a useful engineering scaffold for generating lumen-loaded exosomes directly from producer cells. To test this hypothesis, stable HEK293SF cells were generated to express a codon-optimized full-length Cas9 protein fused to amino acids 1-30 or 1-10 of BASP1 (as described in Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol. February 2015;33(2):139-42). As described above, exosomes were purified from cell cultures and analyzed by SDS-PAGE and Western blotting using an anti-Cas9 antibody (Abcam; catalog number ab191468, clone 7A9-3A3). As Figure 7A shown, both BASP1 1-30 and 1-10 were sufficient to load Cas9 into exosomes. Recombinant Cas9 protein was used as a positive control for Western blotting. Densitometric quantification of the recombinant Cas9 and BASP1-Cas9 exosome lanes from the Western blotting experiment and comparison of various amounts revealed that exosomes loaded 4-5 Cas9 molecules / exosome ( Figure 7B ). Compared to the GFP experiment shown above, the mass of this Cas9 enzyme is approximately 160 kDa, indicating a significant increase in payload size.

[0580] As another validation of the diversity of payload proteins that can be loaded as fusions to the N-terminus of BASP1, ovalbumin was stably expressed in HEK293SF cells as a fusion to amino acids 1-10 of BASP1 (“BASP1(1-10)-OVA”). Using a second selection marker, separate cell lines were co-transfected with the same plasmid and a second plasmid encoding a trimeric CD40L fused to the exosome-specific surface glycoprotein PTGFRN (“3xCD40L-PTGFRN”). Exosomes were purified from both transfected cell cultures and analyzed by SDS-PAGE ( Figure 8A ) and anti-ovalbumin immunoblotting (Abcam; catalog number ab17293, clone 6C8)( Figure 8B) were analyzed. As a control, recombinant ovalbumin (InvivoGen; catalog number vac-pova) was titrated in a separate gel. Ovalbumin was robustly loaded into exosomes when ovalbumin was fused to amino acids 1-10 of BASP1 as a single construct or when combined with an additional overexpression plasmid (3xCD40L-PTGFRN). This result indicates that exosomes can be engineered to combine an endosomal payload and a simultaneous surface payload (e.g., PTGFRN) from a separate transcript.

[0581] Another class of proteins that can be used in the context of therapeutic exosomes are antibodies and antibody fragments. Single-chain camelid nanobodies targeting GFP (as described in Caussinus E, Kanca O, Affolter M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat Struct Mol Biol. December 11, 2011; 19(1):117-21) were stably expressed in HEK293SF cells as fusion proteins with amino acids 1-10 of BASP1 and a tag ("BASP1(1-10)-nanobody") or with a separate ("nanobody") ( Figure 9A ). Purified exosomes were analyzed by SDS-PAGE and Western blotting with an anti- protein antibody, demonstrating that the nanobody was highly enriched in equal amounts of total loaded protein when the nanobody was fused to the N-terminus of BASP1 ( Figure 9B ). These results indicate that different classes of protein payloads can be expressed by producer cells using a very short protein sequence (i.e., a scaffold) derived from the N-terminus of BASP1 and packaged into exosomes.

[0582] Example 3

[0583] The N-terminus of BASP1 can be used to load nucleic acids into the lumen of exosomes

[0584] Nucleic acids, and in particular RNA (e.g., mRNA, siRNA, miRNA), are an attractive class of therapeutic payloads to be loaded into the lumen of therapeutic exosomes. Exosomal loading of RNA can protect the RNA from degradation in the extracellular environment, and the loaded exosomes can be directed to certain cells and / or tissues by additional levels of exosome engineering (e.g., surface expression of targeting constructs). To understand whether exosomes carrying mRNA could be generated using the EV (e.g., exosome) proteins (or protein fragments) identified above, combinatorially engineered exosomes were generated. As Figure 10As shown, amino acids 1-30 of BASP1 are expressed as a fusion with and a variant of the phage protein MCP. MCP recognizes and binds to an mRNA stem-loop called MS2, which can be expressed as a transcriptional fusion with mRNA and other RNAs, driving a physical association between the MCP fusion protein and the target MS2 fusion RNA. Mutation analysis previously identified two positions in MCP that increase its affinity for MS2; the substitution of valine to isoleucine at position 29 (V29I; Lim & Peabody, RNA. Nucleic Acids Res. September 11, 1994; 22(18):3748-52) and the substitution of asparagine to lysine at position 55 (N55K; Lim et al., J Biol Chem. March 25, 1994; 269(12):9006-10). BASP1 1-30 is fused to monomeric or dimeric MCP variants, where each MCP is either V29I or the double mutant V29I / N55K. A luciferase reporter gene construct is expressed as a fusion with 3 MS2 stem-loops from a separate plasmid. Five stable HEK293SF cell lines were generated, either the luciferase-MS2 alone (#811) or in combination with each BASP1-MCP variant (#815, 817, 819 or 821) ( Figure 10 ). As an additional control, HEK293SF cells were stably transfected with FLAG-tagged BASP11-27. Exosomes were isolated and treated with to remove any externally associated mRNA and purified as described above. The purified exosomes were analyzed by SDS-PAGE ( Figure 11A ) and anti- western blotting ( Figure 11B ), demonstrating equal amounts of total protein and comparable levels of BASP1- fusions in each exosome preparation. Importantly, the expression levels of the BASP1-MCP fusions were comparable to those of the BASP1 1-27FLAG fusion lacking the MCP protein, indicating that the addition of MCP monomers or dimers does not disrupt protein loading in BASP1-mediated exosomes.

[0585] Isolate cells stably expressing BASP1-MCP and luciferase-MS2 mRNA, and quantify total luciferase mRNA by RT-qPCR (FWD primer: 5’-TGGAGGTGCTCAAAGAGTTG-3’ (SEQ ID NO:119); REV primer: 5’-TTGGGCGTGCACTTGAT-3’ (SEQ ID NO:120); probe: 5’- / 56-FAM / CAGCTTTCC / ZEN / GGGCATTGGCTTC / 3IABkFQ / -3’ (SEQ ID NO:121)). The luciferase levels expressed by untransfected cells were lower than those of all cells expressing 811, and the latter expressed comparable luciferase levels ( Figure 12A , upper panel above). Purified exosomes from each stable cell line were also analyzed by RT-qPCR. Native exosomes had no detectable levels of luciferase MS2, while cells expressing 811 alone had detectable but very low levels of luciferase MS2. Importantly, each BASP1-MCP fusion protein contained a larger amount of luciferase-MS2 mRNA, demonstrating the importance of the binding between MCP and MS2 in promoting mRNA loading into exosomes ( Figure 12A , lower panel below). Quantification of relative mRNA between groups showed that all BASP1-MCP fusions were enriched approximately 30-60-fold compared to 811 alone ( Figure 12B ). The BASP1-MCP construct 821 containing the dimer MCP V29I / N55K was predicted to have the greatest affinity for MS2 mRNA and indeed contained the largest amount of luciferase-MS2 in this experiment. These results indicate that the BASP1 fragment is a potent and versatile scaffold protein for loading various payloads, including nucleic acids, into the lumen of exosomes.

[0586] Example 4

[0587] BASP1, MARCKS, and MARCKSL1 can be used to generate surface-decorated exosomes

[0588] Results from previous experiments indicated that the full-length and N-terminal regions of MARCKS, MARCKSL1, and BASP1 can be used to generate lumen-loaded exosomes. To further explore the potential of these proteins for exosome engineering, amino acids 1-30 of MARCKS, MARCKSL1, and BASP1 or amino acids 1-10 of BASP1 were fused to the endogenous transmembrane region of CD40L expressed as a homotrimer. Constructs were prepared for the human and mouse sequences of CD40L because the ligand does not cross-react with the homologous receptor on other species ( Figure 13)。Exosomes were purified from HEK293SF cells stably transfected with one of the CD40L expression constructs and incubated in murine or human B cells. The amount of input CD40L on the exosomes was quantified by CD40L ELISA (for measurement of human CD40L, R&D Systems, catalog number DCDL40, lot number P168248; and for measurement of murine CD40L, Abcam, catalog number ab119517, lot number GR3218850-2), B cells were quantified using the B cell marker CD19, and B cell activation was measured by the percentage of gated cells positive for CD69. In species-matched cultures, dose titration curves of exosomal CD40L from mice ( Figure 14A ) or humans ( Figure 14B ) showed comparable activity between constructs on a particle-to-particle basis or when compared to each other (left panel and table below), as well as comparable activity between equimolar recombinant proteins based on CD40L molarity (right panel and table below). Comparable activity was observed when the CD40L constructs were also expressed as monomers and was only slightly weaker than trimeric CD40L expressed on the N-terminus of PTGFRN, a high-density exosome display scaffold (see, e.g., International Patent Application No. PCT / US2018 / 048026) ( Figure 14C ). These results demonstrate that MARCKS, MARCKSL1, and BASP1 are diverse, potent scaffolds that can be used to generate a variety of engineered exosomes for human and animal applications.

[0589] Example 5

[0590] Expression of BASP1, MARCKS, and / or MARCKSL1 in Different Cell Types

[0591] Cell lines from different source tissues (HEK293, kidney; HT1080, connective tissue; K562, bone marrow; MDA-MB-231, breast; Raji, lymphoblast) were grown to logarithmic phase and transferred to medium supplemented with exosome-depleted serum for approximately 6 days, except for HEK293 cells, which were grown in chemically defined medium. Bone marrow-derived mesenchymal stem cells (MSCs) were grown on 3D microcarriers for 5 days and supplemented with serum-free medium for 3 days. The supernatant of each cell line culture was isolated and OPTIPREP as described above was used TMPurify exosomes by density gradient ultracentrifugation. As described above, analyze each purified exosome preparation by LC-MS / MS and quantify the number of peptide spectral matches (PSMs) for BASP1, MARCKS, and MARCKSL1, as well as two widely studied EV (e.g., exosome) proteins (CD81 and CD9). The tetraspanins CD81 and CD9 are detectable in most purified exosome populations, but in some cases, are equal to or lower than luminal EV (e.g., exosome) proteins (e.g., comparing CD9 to BASP1 or MARCKSL1)( Figure 15 ). This finding suggests that the newly identified luminal exosome markers could be suitable fusion proteins for generating engineered exosomes from several unrelated cell lines derived from different tissues.

[0592] Example 6

[0593] Non-human cells overexpressing BASP1 produce luminal engineered exosomes

[0594] The results in Example 5 demonstrated that many human-derived cells naturally express BASP1 and another novel EV. To determine whether BASP1 could be used as a universal exosome scaffold protein, Chinese hamster ovary (CHO) cells were stably transfected with a plasmid expressing full-length BASP1 fused to tag and GFP (“BASP1-GFP-FLAG”), a plasmid expressing amino acids 1-30 of BASP1 fused to tag and GFP (“BASP1(1-30)-GFP-FLAG”), or a plasmid expressing amino acids 1-8 of BASP1 fused to tag and GFP (“BASP1(1-8)-GFP-FLAG”). Exosomes were purified from wild-type CHO cells and CHO cells transfected with one of the three BASP1 plasmids. As Figure 16 A- Figure 16 shown in B, BASP1 and BASP1 fragment fusion proteins were successfully overexpressed in CHO cells and were loaded into exosomes, as detected by stain-free PAGE ( Figure 16 A) and western blotting using an antibody against ( Figure 16 B). This result indicates that non-human cells, such as CHO cells, can produce exosomes overexpressing human BASP1 fragments, and that such overexpression can drive payload proteins into the lumen of exosomes at high density. This result suggests that BASP1 is a universal scaffold protein for generating engineered exosomes from many different cell types and species.

[0595] ***

[0596] It should be understood that the detailed description section, rather than the summary and abstract sections, is intended to explain the claims. The summary and abstract sections may set forth one or more but not all exemplary embodiments of the disclosure as contemplated by one or more of the inventors, and thus are not intended to limit the disclosure and the appended claims in any way.

[0597] The present disclosure has been described above in terms of functional building blocks that specify the implementation of the specified functions and their relationships. For convenience of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries may be defined as long as the specified functions and their relationships are properly implemented.

[0598] Incorporation by reference

[0599] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the extent that each individual publication, patent, patent application, or other document is individually indicated to be incorporated by reference for all purposes.

[0600] Equivalent case

[0601] The present disclosure particularly provides compositions of EVs (e.g., exosomes) containing modified exogenous proteins and peptides for enriching exogenous proteins in EVs (e.g., exosomes). The present disclosure also provides methods for generating enriched EVs (e.g., exosomes). Although various specific embodiments have been shown and described, the above description is not restrictive. The breadth and scope of the present disclosure should not be limited by any of the above exemplary embodiments, but should be defined only by the following claims and their equivalents. It should be understood that various changes can be made without departing from the spirit and scope of the present disclosure. After reading this specification, many variations will be apparent to those skilled in the art.

[0602] This application claims the benefit of International Application No. PCT / US2018 / 061679, filed on November 16, 2018, and U.S. Provisional Application No. 62 / 835,430, filed on April 17, 2019, which are hereby incorporated by reference in their entirety. Sequence listing <110> Kodiak Biosciences, Inc. <120> Engineered Extracellular Vesicles and Their Uses <130> 4000.041PC01 / C-K / BMD <140> PCT / US2018 / 061679 <141> 2018-11-16 <150> US 62 / 835,430 <151> April 17, 2019 <160> 425 <170> PatentIn version 3.5 <210> 1 <211> 332 <212> PRT <213> Artificial Sequence <220> <223> MARCKS protein <400> 1 Met Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala 1 5 10 15 Glu Arg Pro Gly Glu Ala Ala Val Ala Ser Ser Pro Ser Lys Ala Asn 20 25 30 Gly Gln Glu Asn Gly His Val Lys Val Asn Gly Asp Ala Ser Pro Ala 35 40 45 Ala Ala Glu Ser Gly Ala Lys Glu Glu Leu Gln Ala Asn Gly Ser Ala 50 55 60 Pro Ala Ala Asp Lys Glu Glu Pro Ala Ala Ala Gly Ser Gly Ala Ala 65 70 75 80 Ser Pro Ser Ala Ala Glu Lys Gly Glu Pro Ala Ala Ala Ala Ala Pro 85 90 95 Glu Ala Gly Ala Ser Pro Val Glu Lys Glu Ala Pro Ala Glu Gly Glu 100 105 110 Ala Ala Glu Pro Gly Ser Pro Thr Ala Ala Glu Gly Glu Ala Ala Ser 115 120 125 Ala Ala Ser Ser Thr Ser Ser Pro Lys Ala Glu Asp Gly Ala Thr Pro 130 135 140 Ser Pro Ser Asn Glu Thr Pro Lys Lys Lys Lys Lys Arg Phe Ser Phe 145 150 155 160 Lys Lys Ser Phe Lys Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys 165 170 175 Glu Ala Gly Glu Gly Gly Glu Ala Glu Ala Pro Ala Ala Glu Gly Gly 180 185 190 Lys Asp Glu Ala Ala Gly Gly Ala Ala Ala Ala Ala Ala Glu Ala Gly 195 200 205 Ala Ala Ser Gly Glu Gln Ala Ala Ala Pro Gly Glu Glu Ala Ala Ala 210 215 220 Gly Glu Glu Gly Ala Ala Gly Gly Asp Pro Gln Glu Ala Lys Pro Gln 225 230 235 240 Glu Ala Ala Val Ala Pro Glu Lys Pro Pro Ala Ser Asp Glu Thr Lys 245 250 255 Ala Ala Glu Glu Pro Ser Lys Val Glu Glu Lys Lys Ala Glu Glu Ala 260 265 270 Gly Ala Ser Ala Ala Ala Cys Glu Ala Pro Ser Ala Ala Gly Pro Gly 275 280 285 Ala Pro Pro Glu Gln Glu Ala Ala Pro Ala Glu Glu Pro Ala Ala Ala 290 295 300 Ala Ala Ser Ser Ala Cys Ala Ala Pro Ser Gln Glu Ala Gln Pro Glu 305 310 315 320 Cys Ser Pro Glu Ala Pro Pro Ala Glu Ala Ala Glu 325 330 <210> 2 <211> 195 <212> PRT <213> Artificial Sequence <220> <223> MARCKSL1 protein <400> 2 Met Gly Ser Gln Ser Ser Lys Ala Pro Arg Gly Asp Val Thr Ala Glu 1 5 10 15 Glu Ala Ala Gly Ala Ser Pro Ala Lys Ala Asn Gly Gln Glu Asn Gly 20 25 30 His Val Lys Ser Asn Gly Asp Leu Ser Pro Lys Gly Glu Gly Glu Ser 35 40 45 Pro Pro Val Asn Gly Thr Asp Glu Ala Ala Gly Ala Thr Gly Asp Ala 50 55 60 Ile Glu Pro Ala Pro Pro Ser Gln Gly Ala Glu Ala Lys Gly Glu Val 65 70 75 80 Pro Pro Lys Glu Thr Pro Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys 85 90 95 Pro Phe Lys Leu Ser Gly Leu Ser Phe Lys Arg Asn Arg Lys Glu Gly 100 105 110 Gly Gly Asp Ser Ser Ala Ser Ser Pro Thr Glu Glu Glu Gln Glu Gln 115 120 125 Gly Glu Ile Gly Ala Cys Ser Asp Glu Gly Thr Ala Gln Glu Gly Lys 130 135 140 Ala Ala Ala Thr Pro Glu Ser Gln Glu Pro Gln Ala Lys Gly Ala Glu 145 150 155 ...

Claims

1. An isolated extracellular vesicle (EV) comprising a bioactive molecule linked to a scaffold protein, wherein the scaffold protein comprises 7 to 30 amino acids and comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND associates with the luminal surface of the EV and the ED associates with the luminal surface of the EV, wherein the ND comprises the amino acid sequence GGKLSK (SEQ ID NO: 203) but does not comprise methionine (Met) at the N-terminus, wherein the ED comprises lysine (Lys) at its N-terminus, which is directly linked to the lysine at the C-terminus of SEQ ID NO: 203 in the ND; and wherein the bioactive molecule comprises one or more heterologous proteins fused to the C-terminus of the scaffold protein.

2. The EV according to claim 1, wherein the ED comprises (Lys)n, where n is an integer between 2 and 10.

3. The EV according to claim 2, wherein the ED comprises KK, KKK, KKKK (SEQ ID NO: 151), KKKKK (SEQ ID NO: 152) or any combination thereof.

4. The EV according to claim 1, wherein the scaffold protein is linked to the bioactive molecule via a linker.

5. The EV according to claim 4, wherein the linker comprises a cleavable linker.

6. The EV according to any one of claims 1 to 5, wherein the scaffold protein comprises GGKLSKKK (SEQ ID NO: 161) or GGKLSKKS (SEQ ID NO: 162).

7. The EV according to any one of claims 1 - 5, wherein the scaffold protein has a length of at least 8 amino acids.

8. The EV according to any one of claims 1 - 5, wherein the bioactive molecule is on the luminal surface or within the lumen of the EV.

9. The EV according to any one of claims 1 - 5, wherein the scaffold protein further comprises: (i) a transmembrane domain, (ii) an extracellular domain of the vesicle, or (iii) both (i) and (ii).

10. The EV according to claim 9, wherein: (i) the transmembrane domain is between the ED domain of the scaffold protein and the bioactive molecule, and (ii) the bioactive molecule is linked to the extracellular domain of the vesicle.

11. The EV according to any one of claims 1 - 5, wherein the bioactive molecule comprises: (a) a protein; (b) a peptide; (c) a polynucleotide; (d) a compound; (e) a virus; (f) an ionophore; or (g) any combination thereof.

12. The EV according to any one of claims 1 - 5, wherein the EV further comprises a second scaffold protein.

13. The EV according to any one of claims 1 - 5, wherein the bioactive molecule is: (a) an inhibitor of a negative checkpoint regulator or an inhibitor of a binding partner of a negative checkpoint regulator; (b) Immunogenic proteins; (c) Toxins, toxoids, or non-toxic mutants of toxins; (d) Activators of co-stimulatory molecules or activators of binding partners of co-stimulatory molecules.

14. The EV according to any one of claims 1-5, wherein the EV is an exosome.

15. The EV according to any one of claims 1 to 5, wherein the ND is associated with the luminal surface of the EV by myristoylation.

16. The EV according to any one of claims 1 to 5, wherein the ED does not contain a transmembrane domain or a cytoplasmic domain of a virus.

17. The EV according to any one of claims 1 to 5, wherein the amino acid residues at the N-terminus of the scaffold protein are synthetic.

18. The EV according to any one of claims 1 to 5, wherein the ND domain is connected to the ED domain by a linker.

19. The EV according to claim 18, wherein the linker comprises a peptide bond or one or more amino acids.

20. The EV according to claim 11, wherein the protein comprises (i) a recombinant peptide, a natural peptide, a synthetic peptide, an antibody, a fusion protein, or any combination thereof; (ii) an enzyme, a cytokine, a ligand, a receptor, a transcription factor, or a combination thereof; (iii) a T cell receptor (TCR), a T cell co-receptor, a major histocompatibility complex (MHC), a human leukocyte antigen (HLA), or a derivative thereof; (iv) a tumor antigen.

21. The EV according to claim 20, wherein the tumor antigen is selected from the group consisting of: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, alpha-folate receptor, CE7R, IL-3, cancer-testis antigen, MART-1 gp100, and TNF-related apoptosis-inducing ligand.

22. The EV according to claim 11, wherein the virus comprises adeno-associated virus, parvovirus, retrovirus, adenovirus, or any combination thereof.

23. The EV according to claim 12, wherein the second scaffold protein comprises a PTGFRN polypeptide, a BSG polypeptide, an IGSF2 polypeptide, an IGSF3 polypeptide, an IGSF8 polypeptide, an ITGB1 polypeptide, an ITGA4 polypeptide, an SLC3A2 polypeptide, an ATP transporter polypeptide, an aminopeptidase N (ANPEP) polypeptide, an ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1) polypeptide, an enkephalinase (MME) polypeptide, or a neuropilin-1 (NRP1) polypeptide.

24. The EV according to claim 13, wherein the negative checkpoint regulator is selected from the group consisting of: (i) cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), (ii) programmed cell death protein 1 (PD-1), (iii) lymphocyte activation gene 3 (LAG-3), (iv) T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), (v) B and T lymphocyte attenuator (BTLA), (vi) T cell immunoreceptor with Ig and ITIM domains (TIGIT), (vii) T cell-activating V-domain Ig inhibitor (VISTA), (viii) adenosine A2a receptor (A2aR), (ix) killer cell immunoglobulin-like receptor (KIR), (x) indoleamine 2,3-dioxygenase (IDO), (xi) CD20, (xii) CD39, and (xiii) CD73.

25. The EV according to claim 13, wherein (i) the toxin is diphtheria toxin, or (ii) the toxoid is tetanus toxoid.

26. The EV according to claim 13, wherein the positive co-stimulatory molecule is selected from the group consisting of: (i) TNF receptor superfamily members; and (ii) CD28 superfamily co-stimulatory molecules.

27. The EV according to claim 26, wherein the TNF receptor superfamily member is selected from the group consisting of: CD120a, CD120b, CD18, OX40, CD40, Fas receptor, M68, CD27, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, RANK, OCIF, TWEAK receptor, TACI, BAFF receptor, ATAR, CD271, CD269, AITR, TROY, CD358, TRAMP, and XEDAR.

28. The EV according to claim 26, wherein the activator of the positive co-stimulatory molecule is a TNF superfamily member, and the TNF superfamily member is selected from the group consisting of: TNFα, TNF-C, OX40L, CD40L, FasL, LIGHT, TL1A, CD27L, Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand, and EDA-2.

29. The EV according to claim 26, wherein the CD28 superfamily co-stimulatory molecule is selected from ICOS and CD28.

30. The EV according to claim 26, wherein the activator of the positive co-stimulatory molecule is ICOS ligand, CD80, or CD86.

31. A pharmaceutical composition comprising the EV according to any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

32. Use of the EV according to any one of claims 1 to 30 in the manufacture of a medicament for preventing or treating a disease in a subject in need thereof.

33. A cell that produces the EV according to any one of claims 1 to 30.

34. A cell comprising one or more vectors, wherein the vector comprises a nucleic acid sequence encoding the scaffold protein and the bioactive molecule according to any one of claims 1 to 30.

35. A kit comprising the EV according to any one of claims 1 to 30 and instructions for use.

36. A method for manufacturing EV, comprising culturing the cell according to claim 33 or 34 under suitable conditions and obtaining EV.

37. A method for anchoring a bioactive molecule to an extracellular vesicle, comprising linking the bioactive molecule according to any one of claims 1 to 30 to the scaffold protein according to any one of claims 1 to 30.

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