Compositions of engineered exosomes and methods of loading cavity exosome payloads

Abstract

The present invention relates to methods of preparing therapeutic exosomes using proteins newly identified as enriched in exosome body cavities. In particular, the present invention provides methods of localizing therapeutic peptides or proteins in exosomes. The methods involve generating a cavity engineered exosome comprising one or more of a higher concentration of an exosome protein, a modification or fragment of the exosome protein, or a fusion protein of the exosome protein with a therapeutic or cargo protein.

Description

[0001] This application is a divisional application of the patent application with application number 201880072250.2, application date November 16, 2018, and invention title "Compositions of Engineered Exosomes and Methods for Loading Exosomal Payloads".

[0002] Cross - reference to related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application 62 / 587,767, filed November 17, 2017, and U.S. Provisional Patent Application 62 / 634,750, filed February 23, 2018, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

[0004] Sequence listing

[0005] This application includes a sequence listing submitted electronically in ASCII format and hereby incorporated by reference in its entirety. The ASCII copy created on November 15, 2018, is named 41406US_CRF_sequencelisting.txt and is 57,837 bytes in size. Background of the invention

[0006] Exosomes are important mediators of inter - cellular communication. They are also important biomarkers for the diagnosis and prognosis of many diseases, such as cancer. As drug - delivery vehicles, exosomes offer many advantages over traditional drug - delivery methods in many therapeutic areas as a new therapeutic modality.

[0007] A central feature 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 desired therapeutic payloads is currently limited. Exosomes can be loaded by overexpressing the desired therapeutic payload in the producing cells, but the efficiency of this loading is usually limited due to the random localization of the payload to the cellular exosome - processing centers. Alternatively, purified exosomes can be loaded ex vivo, for example, by electroporation. These methods may suffer from problems of low efficiency or be limited to small payloads, such as siRNA. Therefore, suitable methods are needed to produce highly efficient and well - defined loaded exosomes to better realize the therapeutic uses and other applications of exosome - based technologies. Summary of the invention

[0008] One aspect of the present invention relates to a novel method of loading exosomes for therapeutic use. Specifically, the method uses newly identified protein markers from the exosome lumen. Specifically, a set of proteins (e.g., myristoylated alanine-rich protein kinase C substrate (MARCKS); myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1); and brain acid-soluble protein 1 (BASP1)) were identified to be highly enriched in the exosome lumen. In addition, a short sequence at the amino terminus of BASP1 has been shown to be sufficient to direct the efficient loading of fluorescent protein cargo molecules to the same extent as the full-length BASP1 protein. This fragment is less than ten amino acids and represents a significant advancement in the field of engineered exosome loading and allows for the efficient and reproducible loading of any therapeutic protein cargo into the exosome lumen without any additional ex vivo manipulation steps. Loading exosomes using the fusion proteins described herein produces engineered exosomes with significantly higher cargo levels compared to any other genetic engineering method described to date.

[0009] The newly identified protein and peptide sequences from exosomes are used in various embodiments of the present invention. For example, some embodiments involve producing a fusion protein by conjugating an exosomal protein or protein fragment with a therapeutically relevant protein and generating exosomes containing the fusion protein in the exosome lumen. The native full-length protein or bioactive fragment of the therapeutically relevant protein can be transported into the exosome lumen by conjugating it with the protein or protein fragment that enriches exosomes.

[0010] The present invention further relates to generating or using lumen-engineered exosomes that are designed to more efficiently load or to load therapeutically relevant proteins in the exosome lumen. For example, the exosome lumen can be modified to contain a higher concentration of the native full-length exosomal protein and / or a protein that is a fragment or modification of the native exosomal protein in the lumen.

[0011] Some embodiments of the present invention relate to a production cell for producing such lumen-engineered exosomes or a method of generating the production cell. An exogenous polynucleotide can be introduced into the production cell transiently or stably to produce surface-engineered exosomes by the production cell.

[0012] Thus, in one aspect, the present invention provides an exosome comprising a target protein, wherein at least a portion of the target protein is expressed by an exogenous sequence, and the target protein comprises MARCKS, MARCKSL1, BASP1, or a fragment or modification thereof.

[0013] In some embodiments, the target protein is present in the exosome at a higher density than different target proteins in different exosomes, wherein the different target proteins include conventional exosomal proteins or variants thereof. In some embodiments, the conventional exosomal proteins are selected from the group consisting of: CD9, CD63, CD81, PDGFR, GPI-anchored proteins, galectin, LAMP2, LAMP2B, and fragments thereof.

[0014] In some embodiments, the exosomes are produced by cells genetically modified to contain the exogenous sequence, optionally wherein the cells are HEK293 cells.

[0015] In some embodiments, the cells contain a plasmid containing the exogenous sequence.

[0016] In some embodiments, the exogenous sequence is inserted into a genomic locus located 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.

[0017] In some embodiments, the target protein is a fusion protein comprising MARCKS, MARCKSL1, BASP1, or a fragment thereof and a therapeutic peptide.

[0018] In some embodiments, the therapeutic peptide is selected from the group consisting of: natural peptides, recombinant peptides, synthetic peptides, or linkers to therapeutic compounds. In some embodiments, the therapeutic compounds are selected from the group consisting of: nucleotides, amino acids, lipids, carbohydrates, and small molecules. In some embodiments, the therapeutic peptide is an antibody or a fragment or modification thereof. In some embodiments, the therapeutic peptide is an enzyme, ligand, receptor, transcription factor, or a fragment or modification thereof. In some embodiments, the therapeutic peptide is an antimicrobial peptide or a fragment or modification thereof.

[0019] In some embodiments, the exosome further comprises a second target protein, wherein the second target protein comprises MARCKS, MARCKSL1, BASP1, or a fragment thereof. In some embodiments, the exosome further comprises a second target protein, wherein the second target protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment thereof.

[0020] In some embodiments, the target protein comprises a peptide of (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118). In some embodiments, the target protein comprises a peptide of (M)(G)(π)(X)(Φ / π)(π)(+)(+), where each in-bracket position 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 five is not (+) and position six is neither (+) nor (Asp or Glu). In some embodiments, the target protein comprises a peptide of (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), where each in-bracket position 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 five is not (+) and position six is neither (+) nor (Asp or Glu).

[0021] In some embodiments, the target protein comprises a peptide of any one of SEQ ID NOs: 4 - 110. In some embodiments, the target protein comprises a peptide of MGXKLSKKK, where X is any amino acid (SEQ ID NO:116). In some embodiments, the target protein comprises a peptide of SEQ ID NO:110. In some embodiments, the target protein comprises a peptide of SEQ ID NO:13.

[0022] In some embodiments, the target protein further comprises a cargo peptide.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising exosomes and an excipient.

[0024] In some embodiments, the pharmaceutical composition is substantially free of macromolecules, where the macromolecules are selected from nucleic acids, foreign proteins, lipids, carbohydrates, metabolites, and combinations thereof.

[0025] In yet another aspect, the present invention provides a cell population that produces the exosomes provided herein.

[0026] In some embodiments, the cell population comprises an exogenous sequence encoding a target protein, wherein the target 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 target protein, wherein the second target 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 target protein, wherein the second target protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment thereof.

[0027] In some embodiments, the exogenous sequence is inserted into a genomic sequence encoding MARCKS, MARCKSL1, or BASP1, wherein the exogenous sequence and the genomic sequence encode the target protein. In some embodiments, the exogenous sequence is in a plasmid.

[0028] In some embodiments, the exogenous sequence encodes 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 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 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.

[0029] In some embodiments, the exogenous sequence encodes a targeting moiety. In some embodiments, the targeting moiety is specific for an organ, tissue, or cell.

[0030] In some embodiments, the second target protein further comprises a targeting moiety. In some embodiments, the targeting moiety is specific for an organ, tissue, or cell.

[0031] In one aspect, the present invention provides a polypeptide for modifying exosomes, which comprises the following sequences: (i) (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118); (ii) (M)(G)(π)(X)(Φ / π)(π)(+)(+), where each position within 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 five is not (+) and position six is neither (+) nor (Asp or Glu); or (iii) (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), where each position within 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 five is not (+) and position six is neither (+) nor (Asp or Glu).

[0032] In some embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs: 4 - 110. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 13. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 110. In some embodiments, the polypeptide comprises the sequence MGXKLSKKK, where X is any amino acid (SEQ ID NO: 116).

[0033] In some embodiments, the polypeptide is fused to a cargo peptide. In some embodiments, the polypeptide is fused to the N-terminus of the cargo peptide.

[0034] In one aspect, the present invention provides a polynucleotide construct that comprises a coding sequence encoding the polypeptide provided herein. In some embodiments, the coding sequence is codon-optimized.

[0035] In another aspect, the present invention provides a method for preparing engineered exosomes, which comprises the following steps: a. introducing a nucleic acid construct encoding a fusion polypeptide into a cell, the fusion polypeptide comprising (i) a first sequence encoding MARCKS, MARCKSL1, BASP1 or a fragment or modification thereof, and (ii) a second sequence encoding a cargo peptide; b. maintaining the cell under conditions allowing the cell to express the fusion polypeptide; and c. obtaining engineered exosomes containing the fusion polypeptide from the cell.

[0036] In some embodiments, the first sequence comprises the following sequences: (i) (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO:118); (ii) M)(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 five is not (+) and position six is neither (+) nor (Asp or Glu); or (iii) (M)(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 five is not (+) and position six is neither (+) nor (Asp or Glu).

[0037] In some embodiments, the polynucleotide comprises the sequence of any one of SEQ ID NOs: 4 - 110. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 110. In some embodiments, the polynucleotide comprises the sequence of MGXKLSKKK, where X is any amino acid (SEQ ID NO: 116).

[0038] In some embodiments, the fusion polypeptide is present in the lumen of the engineered exosomes at a higher density than the different target proteins in different exosomes, wherein the different target proteins include conventional exosome proteins or variants thereof. In some embodiments, the fusion polypeptide is present at a density more than 2-fold higher than the different target proteins in different exosomes. In some embodiments, the fusion polypeptide is present at a density more than 4-fold, 16-fold, 100-fold, or 10,000-fold higher than the different target proteins in different exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings depict various embodiments of the invention for illustrative purposes only. Those skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods shown herein may be employed without departing from the principles of the invention described herein.

[0040] Figure 1 Optiprep containing the sample after ultracentrifugation is provided TM Image of the density gradient. Bracketed are the top fraction containing exosomes ("top"), the middle fraction containing cell debris ("middle"), and the bottom fraction containing high-density aggregates and cell debris ("bottom").

[0041] Figure 2 is a dot plot showing the proteins (Y-axis) identified from the top fraction of the Optiprep TM ultracentrifugation and the proteins (X-axis) identified from the bottom fraction. Proteins plotted above the dashed line represent exosome-enriched proteins (including MARCKS, MARCHSL1, and BASP1), while proteins below the dashed line represent proteins not specific to exosomes.

[0042] Figure 3 A tryptic peptide coverage map of MARCKS (SEQ ID NO:1) is provided.

[0043] Figure 4 A tryptic peptide coverage map of MARCKSL1 (SEQ ID NO:2) is provided.

[0044] Figure 5 A tryptic peptide coverage map of BASP1 (SEQ ID NO:3) is provided.

[0045] Figure 6A A Western blot picture of the total cell lysate (left) and the purified exosome population (right) collected from HEK293 cells is shown. Figure 6A The Western blot of the gel provided in Figure 6B ), MARCKSL1 ( Figure 6C ), and BASP1 (Figure 6D ) is located in purified exosomes and is not detected in total cell lysates or is detected in cell lysates at significantly lower levels compared to exosomes.

[0046] Figure 7 Shows the fluorescence intensity of purified exosomes containing GFP fused to a MARCKS fragment containing amino acids 1 - 30, CD81, or pDisplay.

[0047] Figure 8 Shows the fluorescence intensity of purified exosomes containing GFP fused to full - length MARCKSL1, a MARCKSL1 fragment containing amino acids 1 - 30, CD81, or pDisplay.

[0048] Figure 9 Shows the fluorescence intensity of purified exosomes containing GFP fused to full - length BASP1, a BASP1 fragment containing amino acids 1 - 30, CD81, or pDisplay.

[0049] Figure 10 Shows a schematic diagram of fusion proteins used to determine the minimum BASP1 N - terminal sequence sufficient to load exosomes (SEQ ID NOs 122 - 134 in order of appearance). The fusion proteins are assigned numbers as provided under "pCB".

[0050] Figure 11 Shows a graph from nanoflow cytometry that measures the fluorescence signal of exosomes engineered to express a BASP1 fragment fused to GFP. The x - axis is based on the numerical numbers assigned to the various fusion proteins as provided in Figure 10 below.

[0051] Figure 12 Shows a picture of a stained protein gel indicating equal loading of exosomes loaded with a BASP1 fragment fused to GFP. The dashed arrow indicates the migration position of the BASP1 fusion protein. The lanes are numbered according to the numerical numbers assigned to the various fusion proteins as provided in Figure 10 below.

[0052] Figure 13 Shows a picture of a protein gel stained with Coomassie blue to label total protein. The dashed arrow indicates the migration position of the BASP1 fusion protein. The lanes are labeled with the numerical numbers assigned to the various fusion proteins as provided in Figure 10 below.

[0053] Figure 14Pictures of anti-FLAG Western blots from purified exosomes containing the BASP1 fragment fused to FLAG and GFP are shown. The lanes are numbered according to the numbers assigned to the various fusion proteins as provided in Figure 10 as follows.

[0054] Figure 15 Pictures of anti-Alix Western blots from purified exosomes containing the BASP1 fragment fused to FLAG and GFP are shown, thus confirming equal protein loading. The lanes are numbered according to the protein sequence numbers shown in Figure 10 as follows.

[0055] Figure 16A The sequences of the fusion proteins containing the BASP1 fragment fused to the FLAG tag and GFP are shown (SEQ ID NOs 135 - 142 in order of appearance). Figure 16B The anti-FLAG Western blot results of purified exosomes from cells stably expressing Figure 16A one of the fusion proteins in

[0056] Figure 17A The sequences of the BASP1 fragment (1 - 30) (SEQ ID NO:4) fused to the FLAG tag and GFP and its modified forms (1 - 30 - S6D, 1 - 30 - S6A, and 1 - 30 - L5Q) are shown (SEQ ID NOs 143 - 145 in order of appearance). Figure 17B The anti-FLAG Western blot results of purified exosomes from cells stably expressing Figure 17A one of the fusion proteins in

[0057] Figure 18 An image of a Coomassie-stained protein gel is shown, where exosome samples were purified from cells stably expressing full-length MARCKSL1, BASP1, or the amino acids 1 - 30 of MARCKS, MARCKSL1, or BASP1, all fused to FALG - GFP. The black arrows on the image indicate the bands corresponding to the fusion proteins.

[0058] Figure 19 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) is shown.

[0059] Figure 20AShows the amino acids 1 - 30 of BASP1 (“BASP1 - 30”) (SEQ ID NO:4) and the sequences of fusion proteins (“MARCKS - MG - PSD”, “MARCKS - MA - PSD”, “MARCKS - MG - PSD - K6S” and “MARCKS - MG - PSD - K6A”) of amino acids 1 - 3 of MARCKS or its modified form fused to the PSD domain of MARCKS or its modified form (SEQ ID NOs 146 - 149 in the order of appearance). Point mutations introduced into the MARCKS sequence are in bold. Figure 20B Shows anti - FLAG Western blot results of purified exosomes from cells stably expressing a fusion protein containing Figure 20A the amino acid sequence and FLAG.

[0060] Figure 21 Shows three different consensus sequences derived from functional studies of MARCKS, MARCKSL1, and BASP1, and the amino acid requirements for each sequence for loading cargo into the exosome lumen (SEQ ID NO:118).

[0061] Figure 22A Shows the total protein (top) and anti - Cas9 Western blot (bottom) of native exosomes or exosomes purified from cells stably expressing Cas9 fused to amino acids 1 - 10 or 1 - 30 of BASP1, as well as a reduced amount of recombinant Cas9. Figure 22B (Top) Shows the standard curve of Cas9 densitometry from Figure 22A the Western blot results. Figure 22B (Bottom) Further provides the amount of Cas9, as a fusion protein conjugated to the 1 - 30 amino acid or 1 - 10 amino acid fragment of BASP1, loaded per purified exosome estimated based on the standard curve.

[0062] Figure 23A Shows the protein gel images of exosomes purified from cells stably transfected with a construct expressing a fusion of the BASP1 N - terminus (amino acids 1 - 10) with ovalbumin (“BASP1(1 - 10) - OVA”) or from cells stably transfected with two constructs, one expressing a fusion of the BASP1 N - terminus (amino acids 1 - 10) with ovalbumin and the other expressing CD40L fused to the transmembrane protein PTGFRN (“BASP1(1 - 10) - OVA; 3XCD40L - PTGFRN”). Figure 23A Further shows the image of a protein gel with a reduced amount of recombinant OVA loaded. Figure 23B Shows the anti - ovalbumin Western blot results of samples from Figure 23A ...

[0063] Figure 24A Shows the sequence of a camel nanobody against GFP fused to amino acids 1 - 10 of BASP1 and a FLAG tag (SEQ ID NO: 150). Figure 24B Shows protein gel and anti - FLAG Western blot results of purified exosomes from cells stably expressing Figure 24A the fusion protein (“BASP1(1 - 10) - nanobody”) or a protein lacking the BASP1 sequence (“nanobody”).

[0064] Figure 25 Shows a schematic of an exosome mRNA loading system that contains (i) BASP1(1 - 30) fused to FLAG 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) luciferase mRNA containing 3x MS2 hairpin loops (“luciferase - MS2 mRNA” or “811”; SEQ ID NO: 115).

[0065] Figure 26A Shows a protein gel of exosomes that contain Figure 25 a combination of the mRNA loading construct described in, luciferase mRNA (811) and various BASP1 fusion proteins (815, 817 or 819). Figure 26B Shows Figure 26A the anti - FLAG Western blot results of the samples in.

[0066] Figure 27A Shows Figure 25 RT - qPCR results of the amount of luciferase mRNA in cells (top) or exosomes (bottom) containing the mRNA loading construct shown in. Figure 27B Shows Figure 27A a table quantifying the amount of luciferase mRNA in purified exosomes from the samples in, which includes fold enrichment relative to random loading of luciferase mRNA.

[0067] Figure 28 Shows a schematic of a CD40L trimer that is fused to the N - terminal fragments of MARCKS, MARCKSL1 and BASP1 to allow outer - surface display of a transmembrane protein anchored in the exosome lumen.

[0068] Figure 29A Shows the results of mouse B cell activation in cultures incubated with exosomes expressing CD40L on the surface, which are fused with the N-terminal fragments of MARCKS, MARCKSL1, and BASP1. Figure 29B Shows the results of human B cell activation in cultures incubated with exosomes expressing CD40L on the surface, which are fused with the N-terminal fragments of MARCKS, MARCKSL1, and BASP1. Figure 29C A graph showing the relative potencies of different CD40L surface-displaying exosomes when fused with the N-terminal sequences of MARCKS, MARCKSL1, BASP1, or full-length PTGFRN.

[0069] Figure 30 Provides the peptide spectral match (PSM) numbers of luminal proteins (MARCKS, MARCKSL1, and BASP1) and conventional exosomal 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).

[0070] Figure 31 Shows protein gels (left) and anti-FLAG Western blots (right) of exosomes from Chinese hamster ovary (CHO) cells alone or from cells overexpressing BASP1 or the N-terminal fragment of BASP1 (1-30 or 1-8) fused to FLAG-GFP. Detailed Description

[0071] Definitions

[0072] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings given to them below.

[0073] As used herein, the term "extracellular vesicle" or "EV" refers to cell-derived vesicles that contain a membrane enclosing an internal space. Extracellular vesicles include all membrane-bound vesicles that have a diameter smaller than that of their source cell. Generally, extracellular vesicles have a diameter ranging from 20 nm to 1000 nm and can contain various macromolecular payloads within the internal space, displayed on the outer surface of the extracellular vesicle, and / or spanning the membrane. The payloads can include 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 successive extrusion or treatment with an alkaline solution), vesicular 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, transplanted tissues or organs, and / or cultured cells.

[0074] As used herein, the term "exosome" refers to small (with a diameter between 20 - 300 nm, more preferably between 40 - 200 nm) cell-derived vesicles that include a membrane enclosing an internal space and are produced by the cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes are a type of extracellular vesicle. Exosomes contain lipids or fatty acids and polypeptides and optionally contain payloads (e.g., therapeutic agents), receptors (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 the producer cells based on their size, density, biochemical parameters, or a combination thereof.

[0075] As used herein, the term "nanovesicle" refers to small (with a diameter between 20 - 250 nm, more preferably between 30 - 150 nm) cell-derived vesicles that include a membrane enclosing an internal space and are produced by the cell by direct or indirect manipulation such that the producer cell would not produce the nanovesicles without such manipulation. Suitable manipulations of the producer cell include, but are not limited to, successive extrusion, treatment with an alkaline solution, sonication, or a combination thereof. In some cases, the production of nanovesicles may result in the disruption of the producer cell. Preferably, the population of nanovesicles is substantially free of vesicles derived from the producer cell by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. Nanovesicles contain lipids or fatty acids and polypeptides and optionally contain payloads (e.g., therapeutic agents), receptors (e.g., targeting moieties), polynucleotides (e.g., nucleic acids, RNA, or DNA), sugars (e.g., monosaccharides, polysaccharides, or glycans), or other molecules. Once nanovesicles are derived from the producer cell according to the manipulation, the nanovesicles can be isolated from the producer cell based on their size, density, biochemical parameters, or a combination thereof.

[0076] As used herein, the term "lumen-engineered exosome" refers to an exosome in which the composition of the internal lumen space is modified. For example, the composition of the lumen, such as proteins, lipids, small molecules, carbohydrates, etc., is modified. The composition can be altered by chemical, physical, or biological methods, or by being produced by cells that have been previously modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering, or by being produced by cells that have been previously modified by genetic engineering.

[0077] As used herein, the term "modifier" of a protein refers to a protein that has at least 15% identity to the non-mutated amino acid sequence of the protein. Modifiers of a protein include fragments or variants of the protein. Further modification of the protein can include chemical or physical modification of the fragment or variant of the protein.

[0078] As used herein, the term "fragment" of a protein refers to a protein that is missing at the N- and / or C-terminus compared to the naturally occurring protein. Preferably, fragments of MARCKS, MARCKSL1, or BASP1 retain the ability to specifically target the exosome lumen. Such fragments are also referred to as "functional fragments". Whether a fragment is a functional fragment in the above sense can be evaluated by any method known in the art for determining the protein content of exosomes, including Western blotting, FACS analysis, and fusion of the fragment with an autofluorescent protein such as GFP. In a particular embodiment, a fragment of MARCKS, MARCKSL1, or BASP1 retains at least 50%, 60%, 70%, 80%, 90%, or 100% of the ability of the naturally occurring MARCKS, MARCKSL1, or BASP1 to specifically target exosomes. In a particular embodiment, the ability of a variant of MARCKS, MARCKSL1, BASP1 or a fragment of MARCKS, MARCKSL1, or BASP1 is at least 70%, 80%, 85%, 90%, 95%, or 99% of the ability of MARCKS, MARCKSL1, and BASP1 to specifically target the exosome lumen, respectively. Such ability can be evaluated, for example, by fluorescently labeled variants, in the assays described in the experimental section.

[0079] As used herein, the term "variant" of a protein refers to a protein that shares a specific amino acid sequence identity with another protein as determined by methods known in the art. Variants of a protein can include substitutions, insertions, deletions, frameshifts, or rearrangements in another protein. In a particular embodiment, the variant is a variant having at least 70% identity with MARCKS, MARCKSL1, BASP1, or a fragment of MARCKS, MARCKSL1, or BASP1. In some embodiments, a variant or fragment variant of MARCKS shares at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with MARCKS according to SEQ ID NO:1 or a functional fragment thereof. In some embodiments, a variant or fragment variant of MARCKSL1 shares at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with MARCKSL1 according to SEQ ID NO:2 or a functional fragment thereof. In some embodiments, a variant or fragment variant of BASP1 shares at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with BASP1 according to SEQ ID NO:3 or a functional fragment thereof. In each of the above cases, it is preferred that the variant or fragment variant retains the ability to specifically target the foreign body cavity.

[0080] Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are described in: 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 73:15237-44 (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 several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and is available on the Internet for use in connection with the sequence analysis programs blastp, blasm, blastx, tblastn, and tblastx. A description of BLAST and how to use the program to determine sequence identity can be accessed on the official website of NCBI (National Center for Biotechnology Information) under the NIH (National Institutes of Health).

[0081] 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 the protein that retains the ability to specifically target the foreign body cavity. In a particular embodiment, the ability of a functional variant of MARCKS, MARCKSL1, BASP1 or a fragment of MARCKS, MARCKSL1, or BASP1 is at least 70%, 80%, 85%, 90%, 95% or 99% of the ability of MARCKS, MARCKSL1, and BASP1 to specifically target the foreign body cavity, respectively.

[0082] As used herein, the term "producing cell" refers to a cell used to produce exosomes. The producing cell can be a cell cultured in vitro or a cell in vivo. Producing cells include, but are not limited to, cells known to be capable of efficiently producing exosomes, such as HEK293 cells, Chinese hamster ovary (CHO) cells, and mesenchymal stem cells (MSC).

[0083] As used herein, the term "target protein" or "target peptide" refers to a protein or peptide that can target an extracellular compartment. The target protein or peptide can be a non-mutated protein that naturally targets an extracellular compartment, or a fragment or modification of such non-mutated protein. The target protein can be a fusion protein that contains a flag tag, a therapeutic peptide, a targeting moiety, or other peptide attached to the non-mutated protein, or a modification or fragment of the non-mutated protein. The target protein can contain modifications such as myristoylation, prenylation, or palmitoylation, or a soluble protein attached to the inner leaflet of the membrane via a linker.

[0084] As used herein, the term "cargo protein" or "cargo peptide" refers to any protein or peptide, or fragment or modification thereof, that can be loaded into an exosome or engineered exosome. The cargo protein or peptide can include a therapeutic peptide or protein that acts on a target (e.g., a target cell) that contacts the exosome. The cargo protein can be a fusion protein that contains the target protein or peptide or fragment or modification thereof as described above such that the cargo fusion protein can target an extracellular compartment.

[0085] As used herein, the term "contaminating protein" refers to a protein that is not associated with an exosome. For example, contaminating proteins include proteins that are not encapsulated within an exosome and are not attached or bound to the exosome membrane.

[0086] As used herein, the terms "isolate", "isolated", and "isolating" or "purify", "purified", and "purifying" and "extracted" and "extracting" are used interchangeably and refer to a preparation state (e.g., multiple known or unknown amounts and / or concentrations) of a desired EV that has undergone one or more purification processes (e.g., selection or enrichment of a desired exosome preparation). In some embodiments, isolation or purification as used herein is the process of removing, in part (e.g., a portion), exosomes from a sample containing producer cells. In some embodiments, the isolated exosome composition has no detectable undesired activity, or alternatively, the level or amount of undesired activity is equal to or lower than an acceptable level or amount. In other embodiments, the isolated exosome composition has an amount and / or concentration of the desired exosomes that is equal to or higher than an acceptable amount and / or concentration. In other embodiments, the isolated exosome composition is enriched compared to the starting material (e.g., the producer cell preparation) from which the composition was obtained. This enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or greater than 99.9999% compared to the starting material. In some embodiments, the isolated exosome preparation is substantially free of residual biological products. In some embodiments, the isolated exosome preparation is 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% free of any contaminating biological substances. Residual biological products may include non-biological substances (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products also means that the exosome composition is free of detectable producer cells and only exosomes are detectable.

[0087] The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" encompasses any agent approved by a regulatory agency of the United States Federal Government or listed in the United States Pharmacopeia for use in animals (including humans), as well as any carrier 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 are included that can be used to prepare pharmaceutical compositions and are generally safe, non-toxic, and desirable.

[0088] As used herein, the term "payload" refers to a therapeutic agent that acts on a target (e.g., a target cell) in contact with an EV. Payloads that can be introduced into exosomes and / or producer cells include 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, dsDNA, lncRNAs, and siRNAs), amino acids (e.g., amino acids containing a detectable moiety or a toxin or disrupting translation), polypeptides (e.g., enzymes), lipids, carbohydrates, viruses and viral particles (e.g., adeno-associated viruses and viral 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).

[0089] 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.).

[0090] The terms "individual", "subject", "host", and "patient" are used interchangeably herein and refer to any mammalian subject, specifically a human, in whom diagnosis, treatment, or therapy is desired. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, and in other embodiments, the subject is a human.

[0091] As used herein, the term "substantially free of" means that a sample containing exosomes contains macromolecules at a mass / volume (m / v) percentage concentration of less than 10%. Some fractions may contain less than 0.001%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% (m / v) of macromolecules.

[0092] As used herein, the term "macromolecule" means nucleic acid, exogenous protein, lipid, carbohydrate, metabolite, or a combination thereof.

[0093] As used herein, the term "conventional exosomal 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, an ATP transporter, or fragments or variants thereof are not conventional exosomal proteins.

[0094] Other interpretation conventions

[0095] Ranges recited herein should be understood as a shorthand for all values within the recited range (including the recited endpoint values). For example, the range 1 to 50 should be understood to include any numerical value, combination of numerical values, or sub-range within 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.

[0096] Unless otherwise specified, reference to a compound having one or more stereocenters refers to each stereoisomer and all combinations of its stereoisomers.

[0097] Exosomal protein

[0098] Some embodiments of the present invention relate to the identification, use, and modification of exosomal proteins that are highly enriched in the exosomal lumen. Such exosomal proteins can be identified by analyzing highly purified exosomes using mass spectrometry or other methods known in the art.

[0099] The proteins include various luminal or membrane proteins enriched on the exosomal membrane, such as transmembrane proteins, integrins, and peripheral proteins. Specifically, the proteins include but are not limited to (1) myristoylated alanine-rich protein kinase C substrate (MARCKS); (2) myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1); and (3) brain acid-soluble protein 1 (BASP1).

[0100] One or more exosome proteins identified herein can be selectively used depending on the production cell, production conditions, purification method, or intended application of the exosomes. Exosome proteins enriched in certain exosome lumens having a specific size range, targeting moiety, charge density, payload, etc. can be identified and used in some embodiments of the present invention. In some embodiments, more than one exosome protein can be used simultaneously or subsequently to produce and isolate therapeutic exosomes.

[0101] Lumen-engineered exosomes

[0102] Another aspect of the present invention relates to the production and use of lumen-engineered exosomes. Lumen-engineered exosomes have an internal space whose composition is modified. For example, the composition of the lumen can be modified by altering the protein, lipid, or glycan content of the lumen.

[0103] In some embodiments, lumen-engineered exosomes are produced by chemical and / or physical methods such as PEG-induced fusion and / or sonication fusion.

[0104] In other embodiments, lumen-engineered exosomes are produced by genetic engineering. Exosomes produced from genetically modified production cells or the progeny of genetically modified cells can contain a modified lumen composition. In some embodiments, lumen-engineered exosomes have a higher or lower density of exosome proteins, or include modifications or fragments of exosome proteins.

[0105] For example, lumen-engineered exosomes can be produced from cells transformed with an exogenous sequence encoding an exosome protein or a modification or fragment of an exosome protein. Exosomes including the protein expressed from the exogenous sequence can include a modified lumen protein composition.

[0106] Various modifications or fragments of exosome proteins can be used in embodiments of the present invention. For example, proteins modified to more effectively target the exosome lumen can be used. Proteins modified to contain the minimal fragment required for specific and effective targeting of the exosome lumen can also be used.

[0107] Fusion proteins having therapeutic activity can also be used. For example, the fusion protein can comprise MARCKS, MARCKSL1, BASP1, or a modification thereof, particularly a fragment or variant thereof, and a therapeutic peptide or cargo protein or peptide. In some embodiments, the fusion protein comprises a fragment of the amino terminus of BASP1.

[0108] Therapeutic peptides are selected from the group consisting of natural peptides, recombinant peptides, synthetic peptides, or linkers to therapeutic compounds. The therapeutic compound can be a nucleotide, amino acid, lipid, carbohydrate, or small molecule. The therapeutic peptide can be an antibody, enzyme, ligand, antigen (e.g., a tumor antigen or an antigen from an infectious agent such as a bacterium, virus, fungus, or protozoan), receptor, antimicrobial peptide, transcription factor, or a fragment or modification thereof. The fusion protein can be presented in the exosome lumen and provide therapeutic activity to the exosome.

[0109] In some embodiments, the 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); zinc finger nuclease (ZFN); recombinase; CRISPR / Cas9 complex, CRISPR / Cpf1 complex, CRISPR / C2c1, C2c2, or C2c3 complex, 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.

[0110] In some embodiments, the 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 end fused to a lumen sequence in the exosome lumen and a second end expressed on the exosome surface. In some embodiments, the transmembrane protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment or variant thereof.

[0111] In some embodiments, the therapeutic peptide is a nucleic acid-binding protein. In some embodiments, the nucleic acid-binding protein is Dicer (an Argonaute protein), TRBP, the MS2 bacteriophage coat protein. In some embodiments, the nucleic acid-binding protein additionally comprises one or more RNA or DNA molecules. In some embodiments, the one or more RNAs are miRNA, siRNA, guide RNA, lincRNA, mRNA, antisense RNA, dsRNA, or a combination thereof.

[0112] In some embodiments, the therapeutic peptide is part of a protein-protein interaction system. In some embodiments, the protein-protein interaction system includes the FRB-FKBP interaction system, e.g., the FRB-FKBP interaction system as described by Banaszynski et al., J Am Chem Soc. April 6, 2005; 127(13):4715-21.

[0113] The fusion protein can target the extracellular space and provide therapeutic activity to exosomes.

[0114] In some embodiments, a fusion protein having a targeting moiety is used. For example, the fusion protein can comprise MARCKS, MARCKSL1, BASP1, or a fragment or modification thereof, and a targeting moiety. The targeting moiety can be used to target exosomes to a specific organ, tissue, or cell for treatment using exosomes. In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof. Antibodies and antigen-binding fragments thereof include whole antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and further include single-chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, murine-human antibodies, murine-primate antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as scFv, (scFv)2, Fab, Fab’, and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. Antibodies and antigen-binding fragments thereof also include bispecific antibodies and multispecific antibodies, provided that they exhibit the desired biological activity or function.

[0115] In some embodiments, a fusion protein comprising a viral protein is used. In some embodiments, the fusion protein comprises a viral capsid or envelope protein. In some embodiments, the fusion protein permits the assembly of intact viruses retained within the exosome lumen.

[0116] In some embodiments, compared to a fusion protein lacking any one of MARCKS, MARCKSL1, BASP1, SEQ ID NO:4-109, or a modifier thereof, particularly a fragment or variant thereof, or compared to a fusion protein comprising a conventional exosome protein, a fusion protein comprising any one of MARCKS, MARCKSL1, BASP1, SEQ ID NO:4-109 or a modifier thereof, particularly a fragment or variant thereof, results in enrichment of the fusion protein in exosomes. In some embodiments, the fusion protein comprising any one of MARCKS, MARCKSL1, BASP1, SEQ ID NO:4-109 or a fragment or modifier thereof comprises 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 each bracketed position 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 five is not (+) and position six is neither (+) nor (Asp or Glu). In some embodiments, the fusion protein comprises a peptide having the sequence (M)(G)(π)(X)(Φ / π)(π)(+)(+), where each bracketed position 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 five is not (+) and position six is neither (+) nor (Asp or Glu). In some embodiments, the conventional exosome protein is selected from the list consisting of CD9, CD63, CD81, PDGFR, GPI-anchored proteins, LAMP2, LAMP2B, and fragments thereof.In some embodiments, the enrichment of a fusion protein comprising any one of MARCKS, MARCKSL1, BASP1, SEQ ID NO: 4-109, or a fragment or modification thereof, in exosomes is >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 higher than that of a fusion protein lacking any one of MARCKS, MARCKSL1, BASP1, SEQ ID NO: 4-109, or a fragment or modification thereof, or than that of a fusion protein comprising a conventional exosomal protein. In some embodiments, the protein sequence of any one of SEQ ID NO: 1-109 is sufficient to load exosomes with the fusion protein.

[0117] In some embodiments, lumen-engineered exosomes comprising a fusion protein containing an exogenous sequence and a newly identified exosomal lumen protein have a higher fusion protein density than similar engineered exosomes comprising an exogenous sequence conjugated to a conventional exosomal protein known in the art (e.g., CD9, CD63, CD81, PDGFR, GPI-anchored protein, lactadherin LAMP2 and LAMP2B, fragments thereof, or peptides binding thereto). In some embodiments, the fusion protein containing the newly identified exosomal protein of the present invention is present in the exosomal lumen at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to the fusion protein in other exosomal lumens similarly modified using conventional exosomal proteins. In some embodiments, the fusion protein containing the newly identified exosomal protein of the present invention is present in the exosomal lumen at a density 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 higher compared to the fusion protein in other exosomal lumens similarly modified using conventional exosomal proteins.

[0118] In some embodiments, the fusion protein comprising MARCKS, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with CD9. In some embodiments, the fusion protein comprising MARCKS, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with CD63. In some embodiments, the fusion protein comprising MARCKS, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with CD81. In some embodiments, the fusion protein comprising MARCKS, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with PDGFR. In some embodiments, the fusion protein comprising MARCKS, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with GPI-anchored proteins. In some embodiments, the fusion protein comprising MARCKS, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with galectin. In some embodiments, the fusion protein comprising MARCKS, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with LAMP2. In some embodiments, the fusion protein comprising MARCKS, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with LAMP2B. In some embodiments, the fusion protein comprising MARCKS, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with conventional proteins.In some embodiments, the fusion proteins comprising MARCKS, variants thereof, fragments, fragment variants, or modifications thereof are present at a density that is 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with variants of conventional exosomal proteins.

[0119] In some embodiments, the fusion protein comprising MARCKSL1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with CD9. In some embodiments, the fusion protein comprising MARCKSL1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with CD63. In some embodiments, the fusion protein comprising MARCKSL1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with CD81. In some embodiments, the fusion protein comprising MARCKSL1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with PDGFR. In some embodiments, the fusion protein comprising MARCKSL1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with GPI-anchored proteins. In some embodiments, the fusion protein comprising MARCKSL1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with galectin. In some embodiments, the fusion protein comprising MARCKSL1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with LAMP2. In some embodiments, the fusion protein comprising MARCKSL1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with LAMP2B. In some embodiments, the fusion protein comprising MARCKSL1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with conventional proteins.In some embodiments, the fusion proteins comprising MARCKSL1, variants, fragments, fragment variants, or modifications thereof are present at a density that is 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes modified similarly with variants of conventional exosomal proteins.

[0120] In some embodiments, the fusion protein comprising BASP1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with CD9. In some embodiments, the fusion protein comprising BASP1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with CD63. In some embodiments, the fusion protein comprising BASP1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with CD81. In some embodiments, the fusion protein comprising BASP1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with PDGFR. In some embodiments, the fusion protein comprising BASP1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with GPI-anchored proteins. In some embodiments, the fusion protein comprising BASP1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with galectin. In some embodiments, the fusion protein comprising BASP1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with LAMP2. In some embodiments, the fusion protein comprising BASP1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with LAMP2B. In some embodiments, the fusion protein comprising BASP1, its variants, fragments, fragment variants, or modifications is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with conventional proteins.In some embodiments, the fusion proteins comprising BASP1, variants, fragments, fragment variants, or modifications thereof are present at a density that is 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes similarly modified with variants of conventional exosomal proteins.

[0121] In some embodiments, compared to exosomes similarly modified with CD9, the fusion proteins comprising any one of SEQ ID NOs: 1-109, their variants, fragments, fragment variants, or modifications are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher. In some embodiments, compared to exosomes similarly modified with CD63, the fusion proteins comprising any one of SEQ ID NOs: 1-109, their variants, fragments, fragment variants, or modifications are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher. In some embodiments, compared to exosomes similarly modified with CD81, the fusion proteins comprising any one of SEQ ID NOs: 1-109, their variants, fragments, fragment variants, or modifications are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher. In some embodiments, compared to exosomes similarly modified with PDGFR, the fusion proteins comprising any one of SEQ ID NOs: 1-109, their variants, fragments, fragment variants, or modifications are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher. In some embodiments, compared to exosomes similarly modified with GPI-anchored proteins, the fusion proteins comprising any one of SEQ ID NOs: 1-109, their variants, fragments, fragment variants, or modifications are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher. In some embodiments, compared to exosomes similarly modified with galectin, the fusion proteins comprising any one of SEQ ID NOs: 1-109, their variants, fragments, fragment variants, or modifications are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher. In some embodiments, compared to exosomes similarly modified with LAMP2, the fusion proteins comprising any one of SEQ ID NOs: 1-109, their variants, fragments, fragment variants, or modifications are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher. In some embodiments, compared to exosomes similarly modified with LAMP2B, the fusion proteins comprising any one of SEQ ID NOs: 1-109, their variants, fragments, fragment variants, or modifications are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher.In some embodiments, the fusion proteins comprising any one of SEQ ID NOs: 1-109, variants, fragments, fragment variants, or modifications thereof are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes modified with conventional protein analogs. In some embodiments, the fusion proteins comprising any one of SEQ ID NOs: 1-109, variants, fragments, fragment variants, or modifications thereof are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher compared to exosomes modified with variants of conventional exosome proteins.

[0122] In some embodiments, the lumen-engineered exosomes described herein exhibit superior characteristics compared to lumen-engineered exosomes known in the art. For example, lumen-engineered exosomes generated using the newly identified exosome proteins provided herein contain a more highly enriched modified protein in their lumen compared to exosomes in the prior art (e.g., those generated using conventional exosome proteins). In addition, compared to lumen-engineered exosomes known in the art, the lumen-engineered exosomes of the present invention can have greater, more specific, or more controllable biological activities. For example, lumen-engineered exosomes comprising a therapeutic or biologically relevant exogenous sequence fused to an exosome protein or fragment thereof described herein (e.g., BASP1 or a fragment thereof) can have more desirable engineered characteristics than fusions with scaffolds known in the art. 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, lactadherin, 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 exosome proteins. Previously, overexpression of exogenous proteins relied on the random or haphazard distribution of exogenous proteins in exosomes to generate lumen-engineered exosomes. This resulted in low levels and unpredictable densities of exogenous proteins in exosomes. Thus, the exosome proteins and fragments thereof described herein provide important advances in new exosome compositions and methods for their preparation.

[0123] The fusion proteins provided herein can comprise MARCKS, MARCKSL1, BASP1, or fragments or variants thereof and an additional peptide. The additional peptide can be attached to the N-terminus or C-terminus of the exosome protein or fragment or variant thereof.

[0124] In some embodiments, the fusion proteins provided herein comprise MARCKS, MARCKSL1, BASP1, or fragments or variants thereof, and two additional peptides. The two additional peptides can be attached to the N-terminus or C-terminus of an exosome protein or a fragment or variant thereof. In some embodiments, one of the two additional peptides is attached to the N-terminus of the exosome protein or a fragment or variant thereof, and the other of the two additional peptides is attached to the C-terminus.

[0125] In some embodiments, the compositions and methods for generating lumen-engineered extracellular vesicles described herein comprise nanovesicles.

[0126] Producer cells for producing lumen-engineered exosomes

[0127] The exosomes of the present invention can be produced from cells grown in vitro or from the body fluids of a subject. When exosomes are produced from an in vitro cell culture, various producer cells, such as HEK293 cells, can be used in the present invention. Other cell types that can be used to generate the lumen-engineered exosomes described herein include, but are not limited to, mesenchymal stem cells, T cells, B cells, dendritic cells, macrophages, and cancer cell lines.

[0128] The producer cells can be genetically modified to contain one or more exogenous sequences to produce lumen-engineered exosomes. The genetically modified producer cells can contain exogenous sequences by transient or stable transformation. The exogenous sequences can be transformed as plasmids. The exogenous sequences can be stably integrated into the genomic sequence of the producer cells at a target site or at a random site. In some embodiments, stable cell lines are generated for the production of lumen-engineered exosomes.

[0129] The exogenous sequences can be inserted into the genomic sequence of the producer cells upstream (5'-end) or downstream (3'-end) of the endogenous sequence encoding the exosome protein. Various methods known in the art can be used to introduce the exogenous sequences into the producer cells. For example, cells modified using various gene editing methods (e.g., methods using homologous recombination, transposon-mediated systems, loxP-Cre systems, CRISPR / Cas9, or TALEN) are within the scope of the present invention.

[0130] The exogenous sequences can include sequences encoding an exosome protein or a modifier or fragment of the exosome protein. Additional copies of the sequence encoding the exosome protein can be introduced to produce lumen-engineered exosomes with a higher density of exosome proteins. Exogenous sequences encoding a modifier or fragment of the exosome protein can be introduced to produce lumen-engineered exosomes containing a modifier or fragment of the exosome protein. Exogenous sequences encoding an affinity tag can be introduced to produce lumen-engineered exosomes containing a fusion protein comprising an affinity tag attached to the exosome protein.

[0131] In some embodiments, the lumen-engineered exosomes have a higher exosomal protein density compared to native exosomes isolated from the same or similar producer cell types. In some embodiments, compared to the native exosomes, the exosomal proteins are present on the lumen-engineered exosomes at a density that is 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000-fold or higher. In some embodiments, compared to the native exosomes, the exosomal proteins are present on the lumen-engineered exosomes at a density that 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 higher. In some embodiments, compared to the unmodified exosomal proteins on the native exosomes, the fusion proteins comprising exosomal proteins are present on the lumen-engineered exosomes at a density that 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 higher. In some embodiments, compared to the unmodified exosomal proteins on the native exosomes, the fragments or variants of exosomal proteins are present on the lumen-engineered exosomes at a density that 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 higher.

[0132] In certain embodiments, MARCKS, a fragment or variant of MARCKS, or a modification thereof is present on the lumen-engineered exosomes at a density that 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 higher compared to unmodified MARCKS on the native exosomes. In some embodiments, MARCKSL1, a fragment or variant of MARCKSL1, or a modification thereof is present on the lumen-engineered exosomes at a density that 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 higher compared to unmodified MARCKSL1 on the native exosomes. In some embodiments, BASP1, a fragment or variant of BASP1, or a modification thereof is present on the lumen-engineered exosomes at a density that 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 higher compared to unmodified BASP1 on the native exosomes.

[0133] 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 specific polypeptide as a payload. In some embodiments, the producer cells are modified to contain two exogenous sequences, one encoding an exosome protein or a modification or fragment of the exosome protein, and the other encoding a payload.

[0134] More specifically, lumen-engineered exosomes can be produced by cells transformed with a sequence encoding one or more 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 1 (MARCKSL1); and (3) brain acid-soluble protein 1 (BASP1). Any of the one or more exosome lumen 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).

[0135] In some embodiments, the one or more exocoelomic proteins are expressed in cells transformed with an exogenous sequence encoding their full-length endogenous form. In some embodiments, such exogenous sequence encodes the MARCKS protein of SEQ ID NO:1. In some embodiments, such exogenous sequence encodes the MARCKSL1 protein of SEQ ID NO:2. In some embodiments, such exogenous sequence encodes the BASP1 protein of SEQ ID NO:3.

[0136] Vesicular engineered exosomes can be produced from cells transformed with a sequence encoding one or more exosomal vesicle protein fragments, the exosomal vesicle proteins including but not limited to (1) myristoylated alanine-rich protein kinase C substrate (MARCKS); (2) myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1); and (3) brain acid soluble protein 1 (BASP1). In some embodiments, the sequence encodes an exosomal vesicle protein fragment that is deleted at least 5, 10, 50, 100, 200, or 300 amino acids from the N-terminus of the native protein. In some embodiments, the sequence encodes an exosomal vesicle protein fragment that is deleted at least 5, 10, 50, 100, 200, or 300 amino acids from the C-terminus of the native protein. In some embodiments, the sequence encodes an exosomal vesicle protein fragment that is deleted at least 5, 10, 50, 100, 200, or 300 amino acids from both the N-terminus and C-terminus of the native protein. In some embodiments, the sequence encodes an exosomal vesicle protein fragment that lacks one or more functional or structural domains of the native protein. In some embodiments, the fusion protein comprises the peptide of SEQ ID NO: 4-109. In some embodiments, the fusion protein comprises the peptide of SEQ ID NO: 13. In some embodiments, the fusion protein comprises a peptide having the sequence MGXKLSKKK, where X is alanine or any other amino acid (SEQ ID NO: 117). In some embodiments, the fusion protein comprises a peptide having the sequence (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), where each bracketed position 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 five is not (+) and position six is neither (+) nor (Asp or Glu). In some embodiments, the fusion protein comprises a peptide having the sequence (M)(G)(π)(X)(Φ / π)(π)(+)(+), where each bracketed position 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 five is not (+) and position six is neither (+) nor (Asp or Glu).

[0137] In some embodiments, lumen-engineered exosomes can be produced by cells transformed with a sequence encoding an exosome protein or a fragment or modification thereof fused to one or more heterologous proteins. In some embodiments, one or more heterologous proteins are fused to the N-terminus of an exosome protein or a modification thereof, particularly a fragment or variant thereof. In some embodiments, one or more heterologous proteins are fused to the C-terminus of an exosome protein or a modification thereof, particularly a fragment or variant thereof. In some embodiments, one or more heterologous proteins are fused to both the N-terminus and the C-terminus of an exosome protein 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.

[0138] In some embodiments, lumen-engineered exosomes are produced by cells transformed with a sequence encoding a polypeptide that is identical or similar to the full-length or a fragment of a native exosome lumen protein, the native exosome lumen protein including, but not limited to, (1) myristoylated alanine-rich C kinase substrate (MARCKS); (2) myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1); and (3) brain acid-soluble protein 1 (BASP1). In some embodiments, the polypeptide is 50% identical to the full-length or a fragment of a native exosome lumen protein, e.g., 50% identical to SEQ ID NOs: 1-3. In some embodiments, the polypeptide is 60% identical to the full-length or a fragment of a native exosome lumen protein, e.g., 60% identical to SEQ ID NOs: 1-3. In some embodiments, the polypeptide is 70% identical to the full-length or a fragment of a native exosome lumen protein, e.g., 70% identical to SEQ ID NOs: 1-3. In some embodiments, the polypeptide is 80% identical to the full-length or a fragment of a native exosome lumen protein, e.g., 80% identical to SEQ ID NOs: 1-3. In some embodiments, the polypeptide is 90% identical to the full-length or a fragment of a native exosome lumen protein, e.g., 90% identical to SEQ ID NOs: 1-3. In some embodiments, the polypeptide is 95% identical to the full-length or a fragment of a native exosome lumen protein, e.g., 95% identical to SEQ ID NOs: 1-3. In some embodiments, the polypeptide is 99% identical to the full-length or a fragment of a native exosome lumen protein, e.g., 99% identical to SEQ ID NOs: 1-3. In some embodiments, the polypeptide is 99.9% identical to the full-length or a fragment of a native exosome lumen protein, e.g., 99.9% identical to SEQ ID NOs: 1-3.

[0139] In some embodiments, the lumen-engineered exosomes produced by cells comprise a polypeptide having a sequence identical or similar to a fragment of brain acid-soluble protein 1 (BASP1). In some embodiments, the polypeptide is 50% identical to the full length or a fragment of BASP1, e.g., 50% identical to SEQ ID NO:4-109. In some embodiments, the polypeptide is 60% identical to the full length or a fragment of BASP1, e.g., 60% identical to SEQ ID NO:4-109. In some embodiments, the polypeptide is 70% identical to the full length or a fragment of BASP1, e.g., 70% identical to SEQ ID NO:4-109. In some embodiments, the polypeptide is 80% identical to the full length or a fragment of BASP1, e.g., 80% identical to SEQ ID NO:4-109. In some embodiments, the polypeptide is 90% identical to the full length or a fragment of BASP1, e.g., 90% identical to SEQ ID NO:4-109. In some embodiments, the polypeptide is 95% identical to the full length or a fragment of BASP1, e.g., 95% identical to SEQ ID NO:4-109. In some embodiments, the polypeptide is 99% identical to the full length or a fragment of BASP1, e.g., 99% identical to SEQ ID NO:4-109. In some embodiments, the polypeptide is 99.9% identical to the full length or a fragment of BASP1, e.g., 99.9% identical to SEQ ID NO:4-109. In some embodiments, the polypeptide is 100% identical to a fragment of BASP1, e.g., 100% identical to SEQ ID NO:4-109.

[0140] Characterization of Exosomes

[0141] In some embodiments, the methods described herein further comprise the step of characterizing the exosomes contained in each collected fraction. In some embodiments, the contents of the exosomes can be extracted for study and characterization. In some embodiments, the 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.

[0142] Measurement of Exosome Content

[0143] Exosomes can include proteins, peptides, RNA, DNA, and lipids. Total RNA can be extracted using an acid-phenol:chloroform extraction method. Then, the RNA can be purified using a glass fiber filter under conditions that recover total RNA containing small RNAs or that separate small RNA species less than 200 nucleotides in length from longer RNA species such as mRNAs. Since the RNA elutes in a small volume, alcohol precipitation steps may not be required to isolate the RNA.

[0144] Exosome compositions can be evaluated by methods known in the art, including but not limited to transcriptomics, sequencing, proteomics, mass spectrometry, or HP-LC.

[0145] A variety of techniques well-known to those skilled in the art (such as quantitative or semi-quantitative RT-PCR, Northern blot analysis, solution hybridization assays) can be used to measure the nucleotide composition (including RNA and DNA) associated with isolated exosomes. In a particular embodiment, the level of at least one RNA is measured by: reverse transcribing the RNA from the exosome composition to provide a set of target oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides with one or more RNA-specific probe oligonucleotides (e.g., a microarray containing RNA-specific probe oligonucleotides) to provide a hybridization profile of the exosome composition, and comparing the exosome composition hybridization profile with the hybridization profile generated from a control sample. Alteration of the signal of at least one RNA in the test sample relative to the control sample indicates the composition of the RNA.

[0146] Additionally, microarrays can be prepared from gene-specific oligonucleotide probes generated from known RNA sequences. The array can contain two different oligonucleotide probes for each RNA, one containing the active mature sequence and the other specific for the precursor of the RNA (e.g., miRNA and pre-miRNA). The array can also contain controls, such as one or more mouse sequences that differ from human orthologs by only a few bases, which can be used as controls for hybridization stringency conditions. tRNA and other RNAs (e.g., rRNA, mRNA) from both species can also be printed on the microchip, providing internal, relatively stable positive controls for specific hybridization. One or more appropriate controls for non-specific hybridization can also be included on the microchip. For this purpose, sequences are selected based on having no homology to any known RNA.

[0147] Microarrays can be fabricated using techniques known in the art. For example, probe oligonucleotides of appropriate length (e.g., 40 nucleotides) are 5'-amine modified at the C6 position and using a commercially available microarray system (e.g., GeneMachine OmniGrid. TM. 100 microarray and Amersham CodeLink. TM) Printed on the activated glass slide. Labeled cDNA oligomers corresponding to the target RNA are prepared by reverse transcribing the target RNA with labeled primers. After the first-strand synthesis, the RNA / DNA hybrid is denatured to degrade the RNA template. Then, under hybridization conditions, such as 6-fold, the labeled target cDNA thus prepared is hybridized with the microarray chip. SSPE / 30% formamide at 25 °C for 18 hours, followed by washing at 0.75-fold. TNT at 37 °C for 40 minutes. Hybridization occurs at the positions on the array where the probe DNA immobilized on the array recognizes the complementary target cDNA in the sample. The labeled target cDNA labels the exact positions where binding occurs on the array, thus allowing for automated detection and quantification. The output consists of a series of hybridization events, thereby indicating the relative abundance of specific cDNA sequences in the exosome preparation and thus the relative abundance of the corresponding complementary RNA. According to one embodiment, the labeled cDNA oligomer is a biotin-labeled cDNA, prepared from a biotin-labeled primer. Then the microarray is processed by directly detecting the biotin-containing transcript using, for example, streptavidin-Alexa647 conjugate and scanned using conventional scanning methods. The image intensity of each spot on the array is proportional to the abundance of the corresponding RNA in the exosomes.

[0148] Data mining work is done by bioinformatics, including scanning the chip, signal acquisition, image processing, normalization, statistical processing and data comparison, and pathway analysis. Thus, microarrays can analyze hundreds or thousands of polynucleotides simultaneously with high-throughput performance. Microarray profiling of mRNA expression has successfully provided valuable data for gene expression studies in basic research. And the technology has been further applied in the pharmaceutical industry and clinical diagnosis. With the acquisition of more and more miRNA data and the accumulation of evidence of the importance of miRNA in gene regulation, microarrays have become a useful technology for high-throughput miRNA research. Analyzing miRNA levels using polynucleotide probes can also be carried out in various physical forms. For example, the use of microtiter plates or automation can be used to facilitate the processing of a large number of test samples.

[0149] Measurement of exosome size

[0150] In some embodiments, the methods described herein include measuring the size of exosomes and / or exosome populations contained in a purified fraction. In some embodiments, the exosome size is measured as the longest measurable dimension. Generally, the longest general dimension of an exosome is also referred to as its diameter.

[0151] The size of exosomes can be measured using various methods known in the art, such as nanoparticle tracking analysis, multi-angle light scattering, single-angle light scattering, size exclusion chromatography, analytical ultracentrifugation, field flow fractionation, laser diffraction, tunable resistive pulse sensing, or dynamic light scattering.

[0152] Exosome size can be measured using dynamic light scattering (DLS) and / or multi-angle light scattering (MALS). Methods for measuring exosome size using DLS and / or MALS are known to those skilled in the art and include nanoparticle tracking analysis (NTA, e.g., using a Malvern Nanosight NS300 nanoparticle tracking device). In a specific embodiment, a Malvern NanoSight NS300 is used to determine exosome size. In some embodiments, the exosomes described herein have a longest dimension of about 20 - 1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosomes described herein have a longest dimension of about 40 - 1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population in which 90% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population in which 95% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population in which 99% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population in which 90% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population in which 95% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population in which 99% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300).

[0153] Exosome size can be measured using tunable resistive pulse sensing (TRPS). In one specific embodiment, the exosome size as measured by TRPS is determined using an iZON qNANO Gold. In some embodiments, the exosomes described herein have a longest dimension of about 20 - 1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosomes described herein have a longest dimension of about 40 - 1000 nm as measured by TRPS (e.g., iZON qNano Gold). In other embodiments, the exosome populations described herein include populations in which 90% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein include populations in which 95% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein include populations in which 99% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein include populations in which 90% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein include populations in which 95% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by TRPS (e.g., using an iZON qNano Gold). In other embodiments, the exosome populations described herein include populations in which 99% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by TRPS (e.g., using an iZON qNano Gold).

[0154] Exosome size can be measured using an electron microscope. In some embodiments, the electron microscopy method used to measure exosome size is transmission electron microscopy. In one specific embodiment, the transmission electron microscope used to measure exosome size is a Tecnai TM G2 Spirit BioTWIN. Methods for measuring exosome size using an electron microscope are well known to those skilled in the art, and any such method can be applied to measure exosome size. In some embodiments, the exosomes described herein have a longest dimension of about 20 - 1000 nm as measured by scanning electron microscopy (e.g., Tecnai TMThe longest dimension of about 20 - 1000 nm as measured by a G2 Spirit BioTWIN scanning electron microscope. In other embodiments, the exosomes described herein have a longest dimension of about 40 - 1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM The longest dimension of about 40 - 1000 nm as measured by a G2 Spirit BioTWIN scanning electron microscope. In other embodiments, the exosome population described herein includes a population in which 90% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM The longest dimension of about 20 - 1000 nm as measured by a G2 Spirit BioTWIN scanning electron microscope. In other embodiments, the exosome population described herein includes a population in which 95% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM The longest dimension of about 20 - 1000 nm as measured by a G2 Spirit BioTWIN scanning electron microscope. In other embodiments, the exosome population described herein includes a population in which 99% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM The longest dimension of about 20 - 1000 nm as measured by a G2 Spirit BioTWIN scanning electron microscope. In other embodiments, the exosome population described herein includes a population in which 90% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM The longest dimension of about 40 - 1000 nm as measured by a G2 Spirit BioTWIN scanning electron microscope. In other embodiments, the exosome population described herein includes a population in which 95% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM The longest dimension of about 40 - 1000 nm as measured by a G2 Spirit BioTWIN scanning electron microscope. In other embodiments, the exosome population described herein includes a population in which 99% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM The longest dimension of about 40 - 1000 nm as measured by a G2 Spirit BioTWIN scanning electron microscope.

[0155] Individual exosome size can be determined on a particle-by-particle basis by nanoparticle flow cytometry. In some embodiments, the nanoparticle flow cytometer is a NanoFCM (NanoFCM, Inc.; Xiamen, China). In some embodiments, the exosomes described herein have a longest dimension of about 20 - 1000 nm as measured by nanoparticle flow cytometry (e.g., using a NanoFCM). In some embodiments, the exosomes described herein have a longest dimension of about 40 - 1000 nm as measured by nanoparticle flow cytometry (e.g., using a NanoFCM). In some embodiments, the exosome population described herein includes a population in which 90% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by nanoparticle flow cytometry (e.g., using a NanoFCM). In some embodiments, the exosome population described herein includes a population in which 95% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by nanoparticle flow cytometry (e.g., using a NanoFCM). In some embodiments, the exosome population described herein includes a population in which 99% of the exosomes have a longest dimension of about 20 - 1000 nm as measured by nanoparticle flow cytometry (e.g., using a NanoFCM). In some embodiments, the exosome population described herein includes a population in which 90% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by nanoparticle flow cytometry (e.g., using a NanoFCM). In some embodiments, the exosome population described herein includes a population in which 95% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by nanoparticle flow cytometry (e.g., using a NanoFCM). In some embodiments, the exosome population described herein includes a population in which 99% of the exosomes have a longest dimension of about 40 - 1000 nm as measured by nanoparticle flow cytometry (e.g., using a NanoFCM).

[0156] Measurement of exosome charge density

[0157] In some embodiments, the methods described herein include measuring the charge density of exosomes and / or exosome populations contained in a purified fraction. In some embodiments, charge density is measured by potentiometric titration, anion exchange, cation exchange, isoelectric focusing, zeta potential, capillary electrophoresis, capillary zone electrophoresis, or gel electrophoresis.

[0158] Measurement of exosome protein density

[0159] In some embodiments, the methods described herein include measuring the density of exosome proteins on the surface of exosomes. The surface density can be calculated or expressed as mass per unit area, number of proteins per unit area, number of molecules per exosome or molecular signal intensity, molar amount of protein, etc. The surface density can be experimentally measured by methods known in the art, e.g., by using biolayer interferometry (BLI), FACS, Western blotting, fluorescence (e.g., GFP-fusion protein) detection, nanoflow cytometry, ELISA, αLISA, and / or densitometry by measuring bands on a protein gel.

[0160] Examples

[0161] The following examples are put forth so as to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the invention and are not intended to limit the scope in which the inventors regard their invention nor are they intended to represent that the following experiments are all or the only experiments performed. While every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should, of course, be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at 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.

[0162] Unless otherwise indicated, the practice of the present invention will employ conventional protein chemistry, biochemistry, recombinant DNA techniques and pharmaceutical methods within the skill in the art. Such techniques are well explained in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 21st edition (Easton, Pennsylvania: Mack Publishing Company, 2005); Carey and Sundberg Advanced Organic Chemistry 3rd edition (Plenum Press) volumes A and B (1992).

[0163] Example 1: Identification of novel exosome proteins

[0164] Collection of exosomes

[0165] After 9 days, exosomes were collected from the supernatant of a high-density suspension culture of HEK293 SF cells. The supernatant was filtered and fractionated by anion exchange chromatography and eluted in a step gradient of sodium chloride. The peak fraction with the highest protein concentration contained exosomes and contaminating cellular components. The peak fraction was isolated and further fractionated by ultracentrifugation on an Optiprep TM density gradient.

[0166] For the Optiprep TM gradient, a 4-layer sterile gradient was prepared in a 12 mL Ultra-Clear (344059) tube in a SW 41Ti rotor with 4 mL of 45% Optiprep TM , 3 mL of 30% Optiprep TM , 2 mL of 22.5% Optiprep TM , 2 mL of 17.5% Optiprep TM and 1 mL of PBS. The exosome fraction was added to the OptiprepTM in the gradient and ultracentrifuged at 200,000 x g for 16 hours at 4 °C to separate the exosome fraction. Ultracentrifugation yields a top fraction known to contain exosomes, a middle fraction containing medium-density cell debris, and a bottom fraction containing high-density aggregates and cell debris ( Figure 1 ). The exosome layer was then gently collected from approximately 3 mL from the top of the tube.

[0167] The exosome fraction was diluted in approximately 32 mL of PBS in a 38.5 mL Ultra-Clear (344058) tube and ultracentrifuged at 133,900 x g for 3 hours at 4 °C to precipitate the purified exosomes. The precipitated exosomes were then resuspended in a minimal volume of PBS (approximately 200 μL) and stored at 4 °C.

[0168] Sample preparation for LC-MS / MS analysis

[0169] To determine proteins specific to exosomes, the top and bottom fractions of the Optiprep TM gradient were analyzed by liquid chromatography-tandem mass spectrometry. Prior to analysis, the total protein concentration of both samples was determined by the bicinchoninic acid (BCA) assay, after which each sample was appropriately diluted to 125 μg / mL in PBS buffer. Next, 50.0 μL of each sample was added to separate 1.5 mL microcentrifuge tubes containing an equal volume of exosome lysis buffer (60 mM Tris, 400 mM GdmCl, 100 mM EDTA, 20 mM TCEP, 1.0% Triton X-100), followed by the transfer of 2.0 μL of 1.0% Triton X-100 solution. All samples were then incubated at 55 °C for 60 minutes.

[0170] Protein precipitation was performed by adding 1250 μL of ethanol at -20 °C. To increase efficiency, the samples were vortexed vigorously and then sonicated in a water bath for 5 minutes. The precipitated material was pelleted by centrifugation at 15,000 g for 5 minutes at room temperature. The supernatant was decanted, and the precipitated material was dried thoroughly using nitrogen. The pellet was resuspended in 30.0 μL of digestion buffer (30 mM Tris, 1.0 M GdmCl, 100 mM EDTA, 50 mM TCEP, pH 8.5), which also reduced disulfide bonds. Free cysteine residues were alkylated by adding 5.0 μL of alkylation solution (375 mM iodoacetamide, 50 mM Tris, pH 8.5) and incubating the resulting solution in the dark at room temperature for at least 30 minutes.

[0171] After incubation, each sample was diluted with 30.0 μL of 50 mM Tris pH 8.5 and proteolytic digestion was initiated by adding 2.0 μg of trypsin. All samples were pooled and then incubated overnight at 37 °C. After incubation, trypsin activity was stopped by adding 5.0 μL of 10% formic acid. Prior to analysis by LC-MS / MS, each sample was desalted using a Pierce C18 spin column. At the end of this process, each sample was dried and reconstituted in 75.0 μL of 95:5 water:acetonitrile with 0.1% formic acid and transferred to an HPLC vial for analysis.

[0172] LC-MS / MS analysis

[0173] Samples were injected into an UltiMate 3000 RSCL nano (Thermo Fisher Scientific) low-flow chromatography system and tryptic peptides were loaded onto an Acclaim PepMap 100 C18 trapping column (75 μm x 2 cm, 3 μm particle size, pore size, Thermo Fisher Scientific) at a flow rate of 2.500 μL / min using the loading mobile phase (MPL: 95% water, 5% acetonitrile, 0.1% formic acid). Peptides were gradient eluted and separated using mobile phase A (MPA: water, 0.1% formic acid) and mobile phase B (MPB: acetonitrile, 0.1% formic acid) at a flow rate of 300 nL / min through an EASY-Spray LC C18 analytical column (75 μm x 25 cm, 2 μm particle size, pore size, Thermo Fisher Scientific). The stepwise gradient used for elution started at 5% MPB, which was held for 15 minutes during the loading process. Then the percentage of MPB was increased from 5% to 17% over 30 minutes, from 17% to 25% over another 45 minutes, and finally from 25% to 40% over 5 minutes. The most hydrophobic species were removed by increasing to 90% MPB over 5 minutes and then holding there for 9 minutes. The total run time of the method was 130 minutes and allowed sufficient time for column re-equilibration. Wash cycles were performed between analytical injections to minimize carryover.

[0174] Mass analysis was performed using a Q Exactive Basic (Thermo Fisher Scientific) mass spectrometer. The precursor ion mass spectra were measured at a resolution of 70,000 in the m / z range of 400 - 1600 Da. Ten of the strongest precursor ions were selected with a collision energy of 27 and fragmented in the HCD cell, and the MS / MS spectra were measured at a resolution of 35,000 in the m / z range of 200 - 2000 Da. Ions with a charge state of 2 - 4 were selected for fragmentation, and the dynamic exclusion time was set to 30 seconds. An exclusion list containing 14 common polysiloxanes was used to minimize false identification of known contaminants.

[0175] Data processing

[0176] First, proteins were identified and quantified (label-free) using Proteome Discoverer software (version 2.1.1.21, Thermo Fisher Scientific) and the Sequest HT algorithm in combination with the Target Decoy PSM validator. The search was performed against the complete Uniprot Homo sapiens (taxonomy 9606 version: 127,783 entries) or Swiss-Prot Homo sapiens (taxonomy 9606 version 2017-05-10: 42,153 entries) reference databases, as well as a custom Uniprot database containing the E1a protein (7 entries). The following search parameters were used: enzyme, trypsin; up to 2 missed cleavages; a minimum peptide length of 6 residues; a precursor mass tolerance of 10 ppm; and a fragment mass tolerance of 0.02 Da. The search also included specific dynamic modifications (oxidation of M; deamidation of N or Q; phosphorylation of S, T or Y; pyroglutamation of the peptide-terminal E; and acetylation of the protein N-terminus) and a static modification (carbamidomethylation of C).

[0177] In the Target Decoy PSM validator, the maximum δCn and both the strict and loose target false discovery rates (FDR) were set to 1 because the data were searched again using Scaffold software (version 4.8.2, Proteome Software Inc.). In Scaffold, the X! Tandem open-source algorithm was also used to search the data to identify proteins using a protein threshold of 99.0%, a minimum of 2 peptides, and a peptide threshold of 95%.

[0178] To determine the identity of novel exosome-specific proteins, a total peptide spectral match (PSM) comparison was performed for the proteins found in the exosome fraction at the top of the Optiprep TM gradient relative to those in the lower fractions. As Figure 2As shown, the correlation between the top fraction proteins (Y-axis) and the bottom fraction proteins (X-axis) is weak. Proteins plotted above the dashed line represent exosome-enriched proteins, while those below the dashed line represent contaminant-enriched proteins. Importantly, many proteins lacking transmembrane domains and highly enriched in the exosome fraction were identified, including (1) myristoylated alanine-rich protein kinase C substrate (MARCKS); (2) myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1); and (3) brain acid-soluble protein 1 (BASP1). As Figures 3 - 5 shown in the tryptic peptide coverage map, mass spectrometry studies led to extensive coverage of MARCKS ( Figure 3 ), MARCKSL1 ( Figure 4 ), and BASP1 ( Figure 5 ). Since none of these proteins were predicted to have transmembrane domains, it was suggested that they were enriched as soluble proteins in the exosome lumen. In summary, these results indicate that there are many luminal proteins enriched in the purified exosome population, which can be used as payload scaffolds for generating engineered exosomes.

[0179] Example 2: Verification of Luminal Protein Expression

[0180] To confirm that the exosome-specific proteins identified in the mass spectrometry studies were highly enriched in the exosome lumen, Western blotting was performed on total cell lysates and purified exosome populations from HEK293 cells. As Figure 6A shown, equal amounts of total protein from the cell lysate (left) and purified exosomes (right) were loaded onto a denaturing polyacrylamide gel. Western blots of MARCKS ( Figure 6B ), MARCKSL1 ( Figure 6C ), and BASP1 ( Figure 6D ) showed that bands representing the new luminal proteins were readily detectable in exosomes but not in the cell lysate, thus demonstrating that these proteins were highly enriched in exosomes and were visually detectable in total exosome lysates. The high expression and enrichment of these luminal proteins in exosomes provide an opportunity to generate lumen-modified exosomes containing heterologous proteins fused at high levels to any of these new proteins.

[0181] Example 3: Verification of Luminal Loading Using New Proteins as Scaffolds

[0182] To confirm the utility of MARCKS, MARCKSL1, and / or BASP1 as lumen-loading scaffolds, each protein was fused to the N-terminus of GFP. Additionally, the first 30 amino acids of each of these proteins were fused to GFP to determine whether shorter protein fragments could drive the loading of engineered exosomes. Exosomes engineered to contain CD81 (a well-established exosome marker) or PDGFR (a transmembrane protein with moderate exosome-loading efficiency) fused to GFP were used as reference standards.

[0183] Stably selected HEK293SF cells containing each expression construct were grown to high density in 200 ml cultures. The supernatants were collected and purified by Optiprep TM density gradient ultracentrifugation as described in Example 1. The resulting GFP-containing exosomes were measured in a Synergy H1 microplate reader in 96-well format. As Figure 7 shown, the first 30 amino acids of MARCKS fused to GFP ("MARCKS(aa 1-30)") were not sufficient to load exosomes at levels higher than CD81-GFP ("CD81") or PDGFR-GFP ("pDisplay"). Similarly, the first 30 amino acids of MARCKSL1 fused to GFP ("MARCKSL1(aa 1-30)") were not sufficient to increase exosome loading compared to CD81-GFP ("CD81"), but the full-length MARCKSL1-GFP fusion ("MARCKSL1") resulted in a significantly higher signal than CD81-GFP( Figure 8 ). In stark contrast, both the full-length BASP1-GFP fusion ("BASP1") and the first 30 amino acids of BASP1 fused to GFP ("BASP1(aa1-30)") resulted in much greater GFP loading compared to CD81-GFP ("CD81") or PDGFR-GFP ("pDisplay")( Figure 9 ). These results indicate that BASP1 (full-length or N-terminus) and full-length MARCKSL1 can be scaffolds suitable for lumenal expression of exosome cargo proteins.

[0184] Example 4: Identification of the Minimal Protein Sequence Sufficient to Load the Lumenal Exosome Payload

[0185] The results in Example 3 indicated that the N-terminal sequence of BASP1 was sufficient to load protein cargo into the exosome lumen. To determine the minimal peptide sequence with this activity, engineered GFP-loading experiments were performed by generating multiple BASP1 truncations fused to the N-terminus of GFP and measuring the extent to which they were loaded into exosomes. Figure 10A series of fusion proteins used in this experiment are shown, indicating fragments and modifications of the BASP1 sequence, the FLAG tag for Western blot detection, the first few amino acids of GFP, and the glycine / serine linker between each region.

[0186] BASP1 has been reported to be myristoylated, which may play a role in its localization to exosome cavities. To test the role of myristoylation in BASP1 loading, glycine-to-alanine point mutations at predicted myristoylation sites were also tested in GFP loading experiments. BASP1 1-30 (pCB 540) contains single mutations at position 2 (sequence pCB 692), position 3 (pCB 693) or double mutations (pCB 694), and was tested with fusion proteins containing various truncations of BASP1 (pCB 683-691).

[0187] HEK293SF cells were transfected and selected in the presence of puromycin to stably express plasmids encoding Figure 10 each sequence in, and exosomes were purified as described in Example 1. GFP fluorescence of the purified exosomes was analyzed by Nano Flow Cytometry (Flow NanoAnlyzer, NanoFCM, Inc.) to determine the degree of GFP loading. As Figure 11 shown in, exosomes from untransfected cells (i.e., lacking GFP) showed very low signals (WT EXO). Exosomes containing BASP1 G2A-GFP (pCB692) or BASP1 G2A / G3A-GFP (pCB 694) showed similar low levels of GFP signal, while BASP1 G3A-GFP (pCB 693) showed a much higher level, indicating that glycine at position two of BASP1 is crucial for loading BASP1 fragments into exosomes, which may be attributed to myristoylation. BASP1 truncations pCB683-689 also showed high levels of GFP signal, while the shorter fragments pCB690-691 were similar to WT EXO. These results suggest that the nine-amino acid fragment pCB689 is sufficient to drive protein cargo into exosome cavities at very high levels.

[0188] To confirm the Figure 11 results shown in, BASP1 fragment-GFP exosomes were analyzed by Western blot. Equal amounts of protein were loaded onto an SDS-PAGE mini- TGX stain-free gel (Bio-Rad, Inc.) to measure total exosome protein ( Figure 12)。At approximately 30 kDa (dashed arrow), the BASP1-GFP fragment could be detected in several lanes of the protein gel. This visualization method relies on the binding of fluorescent molecules in the gel to the tryptophan residues of the protein, but there is only a single tryptophan residue in each BASP1-GFP fusion protein, which may underestimate the abundance of the BASP1 fragment in each lane. To achieve unbiased measurement of BASP1-GFP loading into exosomes, protein gels containing exosome samples were stained with Coomassie Blue (Invitrogen SimplyBlue SafeStain) ( Figure 13 ). The band pattern of the stained gel allowed clear identification of the BASP1-GFP fusion protein (dashed arrow) and confirmed equal amounts of input protein in each sample, correlating with the results shown in Figure 12 .

[0189] Western blotting with an anti-FLAG antibody (M2 monoclonal antibody, Millipore-Sigma) showed equal amounts of BASP1-GFP in pCB540 (amino acids 1-30) and pCB683-689 ( Figure 14 ), further demonstrating the ability of BASP1 to load into exosome cargo. The anti-FLAG signal of the shorter fragment pCB690-691 was significantly reduced or absent. The BASP1G2A-GFP (pCB692) or BASP1 G2A / G3A-GFP (pCB 694) also lacked signal, while BASP1 G3A-GFP (pCB 693) was expressed at a level similar to pCB540. These results are consistent with the Figure 11 nanoflow cytometry data and confirm that pCB689 is sufficient to load protein cargo into exosomes. Western blotting with an antibody against the identified exosomal protein Alix showed equal signals in all samples, indicating that overexpression of BASP1-GFP does not disrupt the expression pattern of endogenous exosomal proteins or otherwise disrupt exosome biogenesis or composition ( Figure 15 ). Collectively, these results demonstrate that a nine-amino acid tag (MGGKLSKKK - SEQ ID NO:13) can be expressed as a fusion with a heterologous protein and drive that protein into the exosome lumen. Additionally, exosome loading requires position two of the sequence, while position three of the sequence is non-essential. Thus, the sequence MGAKLSKKK (SEQ ID NO:110) or more generally, MGXKLSKKK (SEQ ID NO:116) can also be used to load any target protein into the exosome lumen.

[0190] To identify the minimal BASP1 amino acid sequence between the twelve - amino - acid truncation that promotes loading and the six - amino - acid truncation that fails to promote loading as shown above, single truncation mutants of the N - terminus of BASP1 fused to a FLAG - tag and GFP were generated and stably expressed in HEK293SF cells( Figure 16A ). 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 16B ). 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.

[0191] Serine at BASP1 position 6 is highly conserved among species and in MARCKS and MARCKSL1. To determine whether this amino acid is required for cargo loading into exosomes, HEK293SF cells were stably transfected with expression plasmids encoding BASP1 1 - 30 - FLAG - GFP or BASP1 1 - 30 - FLAG - GFP including point mutants, substituting serine six with aspartic acid (S6D; polar charged substitution) or alanine (S6A; small non - polar substitution). Additionally, lysine at position five was mutated to glutamic acid (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. 2016 Dec 1;27(24):3926 - 3936)( Figure 17A ). BASP1 S6D completely abolished GFP loading into exosomes, while S6A did not alter loading. BASP1 L5Q also did not affect luminal loading, indicating that the negative charge at position six disrupts loading, while polar amino acid substitutions at position five are well - tolerated( Figure 17B ).

[0192] The first thirty 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 similar to BASP1, HEK293SF cells were stably transfected with full - length MARCKS and MARCKSL1 proteins or amino acids 1 - 30 fused to FLAG - GFP. Purified exosomes were analyzed by SDS - PAGE and Coomassie staining to determine the extent of loading. Full - length MARCKS and MARCKSL1 were able to load exosomes together with GFP, but amino acids 1 - 30 were less efficient than the full - length proteins, indicating that there are additional structural or sequence features required for loading in the distal regions of the MARCKS and MARCKSL1 proteins( Figure 18) Sequence analysis of MARCKS and MARCKSL1 revealed regions with potential sequence homology to the N-terminus of BASP1. Amino acids 152 - 173 of MARCKS and amino acids 87 - 110 of MARCKSL1 are lysine-rich, interspersed with phenylalanine and serine residues, and are predicted to be phosphorylation site domains (PSD) or effector domains (ED)( Figure 19 ) HEK293SF cells were stably transfected with a plasmid construct (MG-PSD) that fuses amino acids 1 - 3 of MARCKS to the PSD domain. Individual point mutations were generated at the predicted myristoylation site (MA-PSD) and at position six (K6S and K6A) to determine the role of these residues in exosome loading( Figure 20A ) 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 blocks exosome cargo loading and that the PSD of MARCKS can functionally complement the endogenous N-terminal sequence( Figure 20B ) Collectively, these studies allowed the identification of several motifs sufficient to load cargo into exosomes( Figure 21 ).

[0193] The narrowest motif, Motif 1, permits a protein sequence of (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO:118), where each letter or group of letters within parentheses is an amino acid position, and where additionally position five cannot be a positively charged amino acid (K / R / H) and position six cannot be a negatively charged amino acid (D / E). Sub-motifs of Motif 1 include, but are not limited to, the following protein sequences: (M)(G)(G)(K / Q)(L / F / S / Q)(S / A)(K)(K), (M)(G)(A)(K / Q)(L / F / S / Q)(S / A)(K)(K), (M)(G)(S)(K / Q)(L / F / S / Q)(S / A)(K)(K), (M)(G)(G / A / S)(K)(L / F / S / Q)(S / A)(K)(K), (M)(G)(G / A / S)(Q)(L / F / S / Q)(S / A)(K)(K), (M)(G)(G / A / S)(K / Q)(L)(S / A)(K)(K), (M)(G)(G / A / S)(K / Q)(F)(S / A)(K)(K), (M)(G)(G / A / S)(K / Q)(S)(S / A)(K)(K), (M)(G)(G / A / S)(K / Q)(Q)(S / A)(K)(K), (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S)(K)(K), and (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(A)(K)(K), where position five cannot be a positively charged amino acid (K / R / H), and position six cannot be a negatively charged amino acid (D / E).

[0194] A broader motif, Motif 2, can be expressed as (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), where each position within 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 five is not (+) and position six is neither (+) nor (Asp or Glu).

[0195] The most extensive motif, Motif 3, can be expressed as (M)(G)(π)(X)(Φ / π)(π)(+)(+), where each bracketed position 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 five is not (+) and position six 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 cargo into exosomes to the same or comparable extent as full-length BASP1 or a naturally truncated sequence of BASP1. This in-depth analysis of amino acid sequence - structure - function provides new insights into the need to import bio-expressed cargo into exosomes by producer cells.

[0196] Example 5: The N-terminus of BASP1 is sufficient to load different classes of proteins

[0197] The results in Example 4 indicated that the N-terminus of BASP1 could be a useful engineering scaffold for the direct generation of lumen-loaded exosomes from producer cells. To test this hypothesis, stable HEK293SF cells were generated to express 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). Exosomes were purified from cell cultures as described above and analyzed by SDS-PAGE and western blotting using an anti-Cas9 antibody (Abcam; catalog number ab191468, clone 7A9 - 3A3). As Figure 22A 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 and comparison of various amounts of recombinant Cas9 and BASP1-Cas9 exosome lanes from the western blotting experiment showed that the exosomes were loaded with 4 - 5 Cas9 molecules per exosome ( Figure 22B)。Compared to the GFP experiment shown above, the mass of this Cas9 enzyme is approximately 160 kDa, representing a significant increase in cargo size.

[0198] As an additional validation of the diversity of cargo 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 selectable marker, separate cell lines were co-transfected with the same plasmid and a second plasmid encoding 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 23A ) and anti-ovalbumin western blotting (Abcam; catalog number ab17293, clone 6C8) ( Figure 23B ). As a control, recombinant ovalbumin (InvivoGen; catalog number vac-pova) was titrated in a separate gel. Ovalbumin was robustly loaded into exosomes when fused to amino acids 1-10 of BASP1 as a single construct or in combination with other overexpressed plasmids (3xCD40L-PTGFRN). This result demonstrates that exosomes can be combinatorially engineered with luminal cargo and simultaneous surface cargo (such as PTGFRN) from separate transcripts.

[0199] Another class of proteins that may be useful in the context of therapeutic exosomes are antibodies and antibody fragments. A single-chain camelid nanobody 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) was stably expressed in HEK293SF cells as a fusion protein to amino acids 1-10 of BASP1 and a FLAG tag (“BASP1(1-10)-nanobody”) or a FLAG tag alone (“nanobody”) ( Figure 24A ). Purified exosomes were analyzed by SDS-PAGE and anti-FLAG western blotting, showing a significant enrichment of nanobody when the nanobody was fused to the N-terminus of BASP1 at equal amounts of total loaded protein ( Figure 24B ). These results demonstrate that using a very short protein sequence derived from the N-terminus of BASP1, various classes of protein cargo can be expressed by producer cells and packaged into exosomes.

[0200] Example 6: The N-terminus of BASP1 can be used to load nucleic acids in exosome cavities

[0201] Nucleic acids, and in particular RNA (e.g., mRNA, siRNA, miRNA), are a class of attractive therapeutic cargoes to be loaded in therapeutic exosome cavities. Exosomal loading of RNA can protect the RNA from degradation in the extracellular environment and can direct the loaded exosomes to certain cells and / or tissues through additional levels of exosome engineering (e.g., surface expression of targeting constructs). To understand whether the exosomal proteins (or protein fragments) identified above can be used to generate mRNA-loaded exosomes, combinatorially engineered exosomes were generated. As Figure 25 shown, amino acids 1-30 of BASP1 were expressed as a fusion with FLAG and a variant of the bacteriophage 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, thereby driving the physical association between the MCP fusion protein and the target MS2 fusion RNA. Mutation analysis had previously identified two positions in MCP that increased the affinity for MS2; valine was substituted with isoleucine at position 29 (V29I; Lim & Peabody, RNA. Nucleic Acids Res. September 11, 1994; 22(18):3748-52) and asparagine was substituted with lysine at position 55 (N55K; Lim et al., J Biol Chem. March 25, 1994; 269(12):9006-10). BASP1 1-30 was fused to monomeric or dimeric MCP variants, where each MCP was either V29I or the double mutant V29I / N55K. A luciferase reporter gene construct was expressed as a fusion with 3 MS2 stem-loops from a separate plasmid. Five stable HEK293SF cell lines were generated with either luciferase-MS2 alone (#811) or in combination with each BASP1-MCP variant (#815, 817, 819 or 821)( Figure 25 ). As an additional control, HEK293SF cells were stably transfected with FLAG-tagged BASP1 1-27. Exosomes were isolated and treated to remove any externally associated mRNA and purified according to the method above. By SDS-PAGE( Figure 26A ) and anti-FLAG Western blotting( Figure 26B)Analysis of the purified exosomes demonstrated equivalent total protein and comparable BASP1-FLAG fusion levels in each exosome preparation. Importantly, BASP1-MCP fusion protein expressed at levels comparable to the BASP1 1-27FLAG fusion lacking the MCP protein, indicating that addition of MCP monomers or dimers does not disrupt BASP1-mediated protein loading into exosomes.

[0202] Cells stably expressing BASP1-MCP and luciferase-MS2 mRNA were isolated and total luciferase mRNA was quantified 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)). Un-transfected cells expressed lower luciferase levels than all 811-expressing cells, which expressed comparable luciferase levels ( Figure 27A , top). Purified exosomes from each stable cell line were also analyzed by RT-qPCR. Native exosomes did not have detectable levels of luciferase MS2, while cells expressing only 811 had detectable but very low levels of luciferase MS2. Importantly, each BASP1-MCP fusion protein contained a greater amount of luciferase-MS2 mRNA, indicating the importance of the binding between MCP and MS2 in promoting mRNA loading into exosomes ( Figure 27A , bottom). Quantification of relative mRNA between groups showed an enrichment of approximately 30- to 60-fold for all BASP1-MCP fusions compared to 811 alone ( Figure 27B ). 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 greatest amount of luciferase-MS2 in this experiment. These results indicate that the BASP1 fragment is a robust and versatile scaffold protein for loading various cargos, including nucleic acids, into the exosome lumen.

[0203] Example 7: BASP1, MARCKS, and MARCKSL1 can be used to generate surface-decorated exosomes

[0204] 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, as the ligand does not cross-react with the cognate receptor on other species ( Figure 28 ). Exosomes were purified from HEK293SF cells stably transfected with one of the CD40L expression constructs and incubated in mouse or human B cells. The amount of input CD40L on the exosomes was quantified by CD40L ELISA (for measuring human CD40L, R&D Systems, catalog number DCDL40, lot number P168248; and for measuring mouse 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, mouse ( Figure 29A ) or human ( Figure 29B ) exosome CD40L dose-titration curves showed comparable activities between constructs based on particle-to-particle (left panels and tables below) or when compared to each other and to recombinant protein on a CD40L molar equivalent basis (right panels and tables below). Comparable activities were observed when the CD40L construct was also expressed as a monomer, and its potency was only slightly lower than that of trimeric CD40L expressed on the N-terminus of the high-density exosome display scaffold PTGFRN (see, for example, International Patent Application No. PCT / US2018 / 048026) ( Figure 29C ). These results indicate that MARCKS, MARCKSL1, and BASP1 are distinct, robust scaffolds that can be used to generate various classes of engineered exosomes for human and animal applications.

[0205] Example 8: Expression of BASP1, MARCKS, and / or MARCKSL1 in Different Cell Types

[0206] Cell lines from different source tissues (HEK293, kidney; HT1080, connective tissue; K562, bone marrow; MDA-MB-231, breast; Raji, lymphoblast) were grown to the logarithmic phase and transferred to a medium supplemented with exosome-depleted serum for about 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 five days and supplemented in serum-free medium for three days. Supernatants were isolated from each cell line culture, and exosomes were purified using the Optiprep TM density gradient ultracentrifugation method described above. Each purified exosome preparation was analyzed by LC-MS / MS as described above, and the peptide spectrum match numbers (PSMs) for BASP1, MARCKS, and MARCKSL1, as well as two widely studied exosome proteins (CD81 and CD9), were quantified. In most purified exosome populations, the four-transmembrane proteins CD81 and CD9 were detectable, but in some cases, they were equal to or lower than luminal exosome proteins (e.g., comparing CD9 to BASP1 or MARCKSL1)( Figure 30 ). This finding indicates 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.

[0207] Example 9: Non-human cells overexpressing BASP1 produce luminal engineered exosomes

[0208] The results in Example 8 showed that many human-derived cells naturally express BASP1 and the other new exosome proteins identified in Example 1. 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 a FLAG tag and GFP (“BASP1-GFP-FLAG”), a plasmid expressing amino acids 1-30 of BASP1 fused to a FLAG tag and GFP (“BASP1(1-30)-GFP-FLAG”), or a plasmid expressing amino acids 1-8 of BASP1 fused to a FLAG tag and GFP (“BASP1(1-8)-GFP-FLAG”). Using the method described in Example 1, exosomes were purified from wild-type CHO cells and CHO cells transfected with one of the three BASP1 plasmids. As Figure 31As shown, the BASP1 and BASP1 fragment fusion proteins were successfully overexpressed in CHO cells and loaded into exosomes, as detected by Coomassie-stained PAGE and Western blotting with an anti-FLAG antibody. This result indicates that non-human cells (such as CHO cells) can produce exosomes overexpressing the human BASP1 fragment and that such overexpression can drive cargo proteins into the exosome lumen at high density. This result indicates that BASP1 is a universal scaffold protein for generating engineered exosomes from many different cell types and materials.

[0209] Example 10: Generation of lumen-engineered exosomes

[0210] Production cells for generating lumen-engineered exosomes are prepared by introducing an exogenous sequence encoding an exosome protein or a modifier or fragment of an exosome protein. The exosome protein is a fusion protein comprising the BASP1 fragment disclosed in Example 4 above and a cargo protein. A plasmid encoding the exosome protein is transiently transfected to induce high-level expression of the exosome protein in the exosome lumen.

[0211] A polynucleotide encoding an exosome protein, an exosome protein modifier or fragment, or an exogenous sequence encoding a therapeutic peptide, cargo peptide, or targeting moiety is stably transformed into a production cell to generate lumen-engineered exosomes. The exogenous sequence encoding a therapeutic peptide, cargo peptide, or targeting moiety is inserted into the genomic locus encoding the exosome protein to generate a fusion protein comprising the therapeutic peptide or cargo peptide attached to the exosome protein. A polynucleotide encoding a modified exosome protein is knocked into the genomic locus encoding the exosome protein.

[0212] A production cell line is generated by stably transfecting at least two polynucleotides, each encoding an exosome protein, a modifier or fragment of an exosome protein, or an exogenous peptide (such as a targeting moiety, therapeutic peptide). Two or more exogenous sequences are inserted into multiple genomic loci within or near the genomic sequence encoding the exosome protein to generate lumen-engineered exosomes comprising multiple modified exosome proteins. Each of the multiple modified exosome proteins targets the exosome lumen.

[0213] Incorporation by reference

[0214] 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 same extent as if each individual publication, patent, patent application, or other document were specifically and individually indicated to be incorporated by reference for all purposes.

[0215] Equivalent schemes

[0216] The present disclosure particularly provides exosome compositions containing modified exogenous proteins and peptides, which are used to enrich exogenous proteins in exosomes. The present disclosure also provides methods for producing enriched exosomes and methods. Although various specific embodiments have been shown and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the present invention. After reading this specification, many changes will be obvious to those skilled in the art.

Claims

1. An exosome comprising a target protein, wherein at least a portion of the target protein is expressed by an exogenous sequence, and the target protein comprises MARCKS, MARCKSL1, BASP1, or a fragment or modification thereof.

2. The exosome according to claim 1, wherein the target protein is present in the exosome at a higher density than different target proteins in different exosomes, wherein the different target proteins comprise conventional exosomal proteins or variants thereof.

3. The exosome according to claim 2, wherein the conventional exosomal protein is selected from the group consisting of: CD9, CD63, CD81, PDGFR, GPI-anchored protein, galectin, LAMP2, LAMP2B, and fragments thereof.

4. The exosome according to any one of claims 1-3, wherein the exosome is produced by a cell genetically modified to comprise the exogenous sequence.

5. The exosome according to claim 4, wherein the cell comprises a plasmid containing the exogenous sequence.

6. The exosome according to claim 4, wherein the cell comprises the exogenous sequence inserted into the genome of the cell.

7. The exosome according to claim 6, wherein the exogenous sequence is inserted into a genomic locus located 3' or 5' relative to the genomic sequence encoding MARCKS, MARCKSL1, or BASP1.

8. The exosome according to claim 6, wherein the exogenous sequence is inserted into the genomic sequence encoding MARCKS, MARCKSL1, or BASP1.

9. The exosome according to any one of claims 1-8, wherein the target protein is a fusion protein comprising MARCKS, MARCKSL1, BASP1, or a fragment thereof and a therapeutic compound.

10. The exosome according to claim 9, wherein the therapeutic compound is selected from the group consisting of: nucleotides, amino acids, lipids, carbohydrates, and small molecules.

11. The exosome according to any one of claims 1-10, further comprising a second target protein, wherein the second target protein comprises MARCKS, MARCKSL1, BASP1, or a fragment thereof.

12. The exosome according to any one of claims 1-10, further comprising a second target protein, wherein the second target protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment thereof.

13. The exosome according to any one of claims 1-10, wherein the target protein comprises a peptide of (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118).

14. The exosome according to claim 13, wherein the target protein comprises a peptide of any one of SEQ ID NOs: 4-110.

15. The exosome according to claim 13, wherein the target protein comprises a peptide of MGXKLSKKK, wherein X is any amino acid (SEQ ID NO:116).

16. A pharmaceutical composition comprising the exosomes according to any one of claims 1-15 and an excipient.

17. The pharmaceutical composition according to claim 16, which is substantially free of macromolecules, wherein the macromolecules are selected from nucleic acids, foreign proteins, lipids, carbohydrates, metabolites, and combinations thereof.

18. A cell population for producing the exosomes according to any one of claims 1-15.

19. A polypeptide for modifying exosomes, comprising the following sequences: (i) (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K) (SEQ ID NO:118); (ii) (M)(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 five is not (+) and position six is neither (+) nor (Asp or Glu); or (ii) (M)(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 five is not (+) and position six is neither (+) nor (Asp or Glu); or (iii) (M)(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 five is not (+) and position six is neither (+) nor (Asp or Glu).

20. The polypeptide according to claim 19, comprising the sequence of any one of SEQ ID NOs: 4-110.

21. The polypeptide according to claim 19, which comprises the sequence of MGXKLSKKK, wherein X is any amino acid (SEQ ID NO: 116).

22. The polypeptide according to any one of claims 19-21, wherein the polypeptide is fused with a cargo peptide.

23. A polypeptide construct, which comprises a coding sequence encoding the polypeptide according to any one of claims 19-22.

24. The polypeptide construct according to claim 23, wherein the coding sequence is codon-optimized.

25. A method for preparing engineered exosomes, which comprises the following steps: a. Introducing a nucleic acid construct encoding a fusion polypeptide into a cell, the fusion polypeptide comprising (i) a first sequence encoding MARCKS, MARCKSL1, BASP1 or a fragment or modification thereof, and (ii) a second sequence encoding a cargo peptide; b. Maintaining the cell under conditions allowing the cell to express the fusion polypeptide; and c. Obtaining the engineered exosomes containing the fusion polypeptide from the cell.

26. The method according to claim 25, wherein the first sequence comprises the following sequences: (i) (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118); (ii) (M)(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 five is not (+) and position six is neither (+) nor (Asp or Glu); or (ii) (M)(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 five is not (+) and position six is neither (+) nor (Asp or Glu); or (iii) (M)(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 five is not (+) and position six is neither (+) nor (Asp or Glu).

27. The method according to claim 25 or 26, wherein the first sequence comprises any one of SEQ ID NOs: 4 - 110.

28. The method according to claim 26, wherein the first sequence comprises MGXKLSKKK, where X is any amino acid (SEQ ID NO:116).

29. The method according to any one of claims 25-28, wherein the fusion polypeptide is present in the lumen of the engineered exosome at a higher density than the different target proteins in different exosomes, wherein the different target proteins include conventional exosomal proteins or variants thereof.

30. The method according to claim 29, wherein the fusion polypeptide is present at a density more than 2-fold higher than the different target proteins in the different exosomes.

Images