Compositions of engineered exosomes and methods of loading lumen exosome payloads

Abstract

The present invention relates to methods for preparing therapeutic exosomes using proteins newly identified as being enriched in the lumen of exosomes. Specifically, the present invention provides methods for localizing therapeutic peptides or proteins in exosomes. The methods involve generating lumen-engineered exosomes containing one or more of an exosomal protein, a modification or fragment of the exosomal protein, or a fusion protein of the exosomal protein with a therapeutic or cargo protein at a higher concentration.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 587,767, filed November 17, 2017, and U.S. Provisional Patent Application No. 62 / 634,750, filed February 23, 2018, the disclosures of which are hereby incorporated in their entirety for all purposes.

[0003] Sequence Listing

[0004] 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 Art

[0005] Exosomes are important mediators of intercellular communication. They are also important biomarkers for the diagnosis and prognosis of many diseases (such as cancer). As drug delivery vehicles, exosomes, as a new treatment modality, offer many advantages over traditional drug delivery methods in many therapeutic areas.

[0006] The central feature of allosomes is that they can contain bioactive payloads in their inner space or cavity. As is well known, allosomes contain endogenous payloads (including mRNA, miRNA, DNA, proteins, carbohydrates and lipids), but the ability to guide the specific load of required therapeutic payloads is currently limited. Allosomes can be loaded by overexpressing the required therapeutic payload in production cells, but due to the random positioning of payloads to the cell exosome processing center, the efficiency of the load is generally limited. Alternatively, purified allosomes can be loaded in vitro by, for example, electroporation. These methods may suffer from inefficient problems or be limited to small payloads, such as siRNA. Therefore, suitable methods are needed to produce highly effective and well-defined load allosomes, to better realize the therapeutic use and other applications of allosome-based technology. Summary of the invention

[0007] One aspect of the present invention relates to a new method for loading exosomes for therapeutic use. Specifically, the method uses a newly identified protein marker from the exosome cavity. Specifically, a group 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)) are identified to be highly enriched in the exosome cavity. In addition, a short sequence at the amino terminus of BASP1 has been shown to be sufficient to guide 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, presents a significant advance in the field of engineered exosome loading, and allows any therapeutic protein cargo to be effectively and reproducibly loaded into the exosome cavity without any additional in vitro manipulation steps. Compared with any other genetic engineering method described to date, the use of the fusion protein described herein to load exosomes produces engineered exosomes with significantly higher cargo levels.

[0008] The newly identified protein and peptide sequences from exosomes are used in various embodiments of the present invention. For example, some embodiments involve producing fusion proteins by conjugating exosome proteins or protein fragments with therapeutically relevant proteins and producing exosomes containing the fusion proteins in the exosome cavity. The native full-length protein or biologically active fragment of the therapeutically relevant protein can be transported to the exosome cavity by conjugating it to the protein or protein fragment enriched in the exosome.

[0009] The present invention further relates to the production or use of cavity engineered exosomes, which are designed to be more effectively loaded in the exosome cavity, or to be loaded with therapeutically relevant proteins. For example, the exosome cavity can be modified to contain higher concentrations of natural full-length exosome proteins and / or fragments or modifications of natural exosome proteins in the cavity.

[0010] Some embodiments of the present invention relate to a production cell for producing such a cavity-engineered exosome or a method of producing the production cell.Exogenous polynucleotides can be introduced into the production cell temporarily or stably to produce the surface-engineered exosome by the production cell.

[0011] Therefore, 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.

[0012] In some embodiments, the target protein is present in the exosome at a higher density than a different target protein in a different exosome, wherein the different target protein comprises a conventional exosomal protein or a variant thereof. In some embodiments, the conventional exosomal protein is selected from the group consisting of CD9, CD63, CD81, PDGFR, GPI anchor protein, lactadherin, LAMP2, LAMP2B and fragments thereof.

[0013] In some embodiments, the exosomes are produced by cells that are genetically modified to contain the exogenous sequence, optionally wherein the cells are HEK293 cells.

[0014] In some embodiments, the cell comprises a plasmid comprising an exogenous sequence.

[0015] In some embodiments, the exogenous sequence is inserted into a genomic site 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.

[0016] In some embodiments, the target protein is a fusion protein comprising MARCKS, MARCKSL1, BASP1, or a fragment thereof and a therapeutic peptide.

[0017] In some embodiments, the therapeutic peptide is selected from the group consisting of: natural peptides, recombinant peptides, synthetic peptides or to a joint of a therapeutic compound. In some embodiments, the therapeutic compound is 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 modifier thereof. In some embodiments, the therapeutic peptide is an enzyme, a ligand, a receptor, a transcription factor or a fragment or modifier thereof. In some embodiments, the therapeutic peptide is an antimicrobial peptide or a fragment or modifier thereof.

[0018] 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, ATP transporter, or a fragment thereof.

[0019] 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)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position 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)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is ​​neither (+) nor (Asp or Glu).

[0020] 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, wherein 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.

[0021] In some embodiments, the target protein further comprises a cargo peptide.

[0022] In another aspect, the present invention provides a pharmaceutical composition comprising exosomes and an excipient.

[0023] In some embodiments, the pharmaceutical composition is substantially free of macromolecules, wherein the macromolecules are selected from the group consisting of nucleic acids, exogenous proteins, lipids, carbohydrates, metabolites, and combinations thereof.

[0024] In yet another aspect, the present invention provides a cell population that produces the exosomes provided herein.

[0025] 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, ATP transporter, or a fragment thereof.

[0026] 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.

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

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

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

[0030] In one aspect, the present invention provides a polypeptide for modifying an exosome, comprising the following sequence: (i) (M) (G) (G / A / S) (K / Q) (L / F / S / Q) (S / A) (K) (SEQ ID NO: 118); (ii) (M) (G) (π) (X) (Φ / π) (π) (+) (+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is ​​neither (+) nor (Asp or Glu); or (iii) (M) (G) (π) (ξ) (Φ / π) )(S / A / G / N)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is ​​neither (+) nor (Asp or Glu).

[0031] 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 of MGXKLSKKK, wherein X is any amino acid (SEQ ID NO: 116).

[0032] In some embodiments, the polypeptide is fused to the cargo peptide. In some embodiments, the polypeptide is fused to the N-terminus of the cargo peptide.

[0033] In one aspect, the present invention provides a polynucleotide construct comprising a coding sequence encoding a polypeptide provided herein. In some embodiments, the coding sequence is codon optimized.

[0034] In another aspect, the present invention provides a method for preparing engineered exosomes, comprising the following steps: a. introducing into a cell a nucleic acid construct encoding a fusion polypeptide, wherein the fusion polypeptide comprises (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 an engineered exosome comprising the fusion polypeptide from the cell.

[0035] In some embodiments, the first sequence comprises the following sequence: (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) (+) (+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is ​​neither (+) nor (Asp or Glu); or (iii) (M) (G) (π) (ξ) (Φ / π) (S / A / G / N)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is ​​neither (+) nor (Asp or Glu).

[0036] 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, wherein X is any amino acid (SEQ ID NO: 116).

[0037] In some embodiments, the fusion polypeptide is present in the cavity of the engineered exosome 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 greater than 2 times higher than the different target proteins in different exosomes. In some embodiments, the fusion polypeptide is present at a density greater than 4 times, 16 times, 100 times, or 10,000 times higher than the different target proteins in different exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings depict various embodiments of the present invention for purposes of illustration 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 present invention as described herein.

[0039] Figure 1 Provides Optiprep containing samples after ultracentrifugation TM Image of density gradient. The bracketed fractions 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").

[0040] Figure 2 It is shown that from Optiprep TM Dot plot of proteins identified from the top fraction of ultracentrifugation (Y-axis) and proteins identified from the bottom fraction (X-axis). Proteins plotted above the dashed line represent exosome-enriched proteins (including MARCKS, MARCHSL1, and BASP1), while proteins below the dashed line represent proteins that are not specific to exosomes.

[0041] Figure 3 A tryptic peptide coverage of MARCKS (SEQ ID NO: 1) is provided.

[0042] Figure 4 A tryptic peptide coverage of MARCKSL1 (SEQ ID NO: 2) is provided.

[0043] Figure 5 A tryptic peptide coverage of BASP1 (SEQ ID NO: 3) is provided.

[0044] Fig. 6A Western blot images of total cell lysates (left) and purified exosome populations (right) collected from HEK293 cells are shown. Fig. 6A Western blot of the gel presented in the present invention shows that MARCKS ( Figure 6B )、MARCKSL1( Figure 6C ) and BASP1( Fig.6D ) were located in purified exosomes and were not detected in total cell lysates or were detected in cell lysates at significantly lower levels compared to exosomes.

[0045] Figure 7 Shown are the fluorescence intensities of purified exosomes containing GFP fused to a MARCKS fragment containing amino acids 1-30, CD81, or pDisplay.

[0046] Figure 8 Shown are the fluorescence intensities of purified exosomes containing GFP fused to full-length MARCKSL1, a MARCKSL1 fragment containing amino acids 1-30, CD81, or pDisplay.

[0047] Fig. 9 Shown are the fluorescence intensities of purified exosomes containing GFP fused to full-length BASP1, a fragment of BASP1 containing amino acids 1-30, CD81, or pDisplay.

[0048] Fig.10 A schematic diagram of fusion proteins used to determine the minimal BASP1 N-terminal sequence sufficient for loading exosomes is shown (SEQ ID NOs 122-134, respectively, in order of appearance). The fusion proteins are assigned numbers as provided under "pCB".

[0049] Fig.11 Figure 2 shows a graph from nanoflow cytometry measuring the fluorescent signal of exosomes engineered to express a BASP1 fragment fused to GFP. The x-axis is based on the values ​​assigned to Fig.10 The various fusion proteins are numbered as provided in .

[0050] Fig.12 A picture of a stained protein gel is shown, indicating equal loading of exosomes loaded with BASP1 fragments fused to GFP. The dashed arrows indicate the migration position of the BASP1 fusion protein. The lanes are assigned to Fig.10 The various fusion proteins are numbered as provided in .

[0051] Fig.13 A picture of a protein gel stained with Coomassie blue to mark total protein is shown. The dashed arrows indicate the migration position of the BASP1 fusion protein. The lanes are assigned Fig.10 Numerical labels for various fusion proteins are provided in .

[0052] Fig.14Shown is a picture of an anti-FLAG western blot from purified exosomes containing a BASP1 fragment fused to FLAG and GFP. Lanes are assigned to Fig.10 The various fusion proteins are numerically numbered as provided in .

[0053] Fig.15 Shown is a picture of an anti-Alix western blot from purified exosomes containing BASP1 fragments fused to FLAG and GFP, demonstrating equal protein loading. Lanes are according to Fig.10 The protein sequence numbers are shown in .

[0054] Fig.16A The sequences of fusion proteins comprising a BASP1 fragment fused to a FLAG tag and GFP are shown (SEQ ID NOs 135-142, respectively, in order of appearance). Fig. 16B Shown is the stable expression Fig.16A Anti-FLAG western blot results of purified exosomes from cells expressing one of the fusion proteins.

[0055] Fig.17A The sequences from the BASP1 fragment (1-30) (SEQ ID NO: 4) and its modifications (1-30-S6D, 1-30-S6A and 1-30-L5Q) fused to a FLAG tag and GFP are shown (SEQ ID NOs 143-145, respectively, in order of appearance). Fig. 17B Shown is the stable expression Fig.17A Anti-FLAG western blot results of purified exosomes from cells expressing one of the fusion proteins.

[0056] Fig.18 Shown is an image of a Coomassie-stained protein gel in which exosome samples were purified from cells stably expressing full-length MARCKSL1, BASP1, or 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.

[0057] Fig.19 Shown is 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).

[0058] Fig. 20AThe sequences of amino acids 1-30 of BASP1 ("BASP1-30") (SEQ ID NO: 4) and fusion proteins comprising amino acids 1-3 of MARCKS or a modification thereof fused to the PSD domain of MARCKS or a modification thereof ("MARCKS-MG-PSD", "MARC KS-MA-PSD", "MARCKS-MG-PSD-K6S" and "MARCKS-MG-PSD-K6A") are shown (SEQ ID NOs 146-149, respectively, in order of appearance). Point mutations introduced into the MARCKS sequence are in bold. Fig. 20B Shown are stable expression of Fig. 20A The amino acid sequence and anti-FLAG western blot results of purified exosomes of cells with FLAG fusion protein.

[0059] Fig.21 Three different consensus sequences derived from functional studies of MARCKS, MARCKSL1, and BASP1 are shown, along with the amino acid requirements of each sequence for loading cargo into the exosome lumen (SEQ ID NO: 118).

[0060] Fig.22A Shown are total protein (top) and anti-Cas9 Western blots (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 decreasing amounts of recombinant Cas9. Fig. 22B (Top) shows the Fig.22A Western blot results for Cas9 densitometry standard curve. Fig. 22B (Bottom) Further provided is the amount of Cas9 loaded per purified exosome as a fusion protein conjugated to a 1-30 amino acid or a 1-10 amino acid fragment of BASP1, estimated based on the standard curve.

[0061] Fig.23A Shown are images of protein gels of exosomes purified from cells stably transfected with a construct expressing a fusion of the N terminus of BASP1 (amino acids 1-10) with ovalbumin ("BASP1 (1-10)-OVA") or with two constructs, one expressing a fusion of the N terminus of BASP1 (amino acids 1-10) with ovalbumin and the other expressing CD40L fused to the transmembrane protein PTGFRN ("BASP1 (1-10)-OVA; 3XCD40L-PTGFRN"). Fig.23A Further shown are images of protein gels loaded with decreasing amounts of recombinant OVA. Fig. 23B Shown from Fig.23A Anti-ovalbumin Western blot results of samples.

[0062] Fig.24A The sequence of a camelid nanobody against GFP fused to amino acids 1-10 of BASP1 and a FLAG tag is shown (SEQ ID NO: 150). Fig. 24B Shown are stable expression Fig.24A Protein gel and anti-FLAG western blot results of purified exosomes from cells expressing fusion proteins of BASP1(1-10) ("BASP1(1-10)-Nanobody") or proteins lacking the BASP1 sequence ("Nanobody").

[0063] Fig.25 A schematic diagram of an exosomal mRNA loading system is shown, which contains (i) BASP1 (1-30) fused to FLAG and a monomeric or dimeric MCP variant (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 a 3x MS2 hairpin loop ("luciferase-MS2 mRNA" or "811"; SEQ ID NO: 115).

[0064] Fig.26A A protein gel of exosomes is shown, which contain Fig.25 Combinations of the mRNA loading construct described in , luciferase mRNA (811) and various BASP1 fusion proteins (815, 817 or 819). Fig.26B Shows Fig.26A Anti-FLAG western blot results of samples in.

[0065] Fig.27A Shows that it contains Fig.25 RT-qPCR results of the amount of luciferase mRNA in cells (top) or exosomes (bottom) loaded with the mRNA constructs shown in . Fig.27B The quantitative Fig.27A Table of the amounts of luciferase mRNA in purified exosomes of the samples, including the fold enrichment relative to the random loading of luciferase mRNA.

[0066] Fig.28 Schematic representation of the CD40L trimer fused to MARCKS, MARCKSL1 and the N-terminal fragment of BASP1 to allow external surface display of the transmembrane protein anchored in the exosome lumen.

[0067] Fig.29A Shown are the results of mouse B cell activation in culture incubated with CD40L surface-expressing exosomes fused to the N-terminal fragment of MARCKS, MARCKSL1, and BASP1. Fig.29B Shown are the results of human B cell activation in culture incubated with CD40L surface-expressing exosomes fused to the N-terminal fragment of MARCKS, MARCKSL1, and BASP1. Fig.29C Graph showing the relative potency of different CD40L surface displayed exosomes when fused to the N-terminal sequence of MARCKS, MARCKSL1, BASP1 or full length PTGFRN.

[0068] Fig.30 The number of peptide spectrum matches (PSMs) for luminal proteins (MARCKS, MARCKSL1, and BASP 1) and conventional exosomal proteins (CD81 and CD9) in exosomes purified from various cell lines of different origin (HEK293SF, kidney; HT1080, connective tissue; K562, bone marrow; MDA-MB-231, breast; Raji, lymphoblastoid; mesenchymal stem cells (MSC), bone marrow) are provided.

[0069] Fig.31 Shown are protein gels (left) and anti-FLAG Western blots (right) of Chinese hamster ovary (CHO) cell-derived exosomes alone, or exosomes from cells overexpressing BASP1 or BASP1 N-terminal fragments (1-30 or 1-8) fused to FLAG-GFP. DETAILED DESCRIPTION

[0070] definition

[0071] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, the following terms have the meanings ascribed thereto below.

[0072] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle that includes a membrane that encloses an internal space. Extracellular vesicles include all membrane-bound vesicles whose diameter is less than the diameter of their source cells. Typically, the diameter of an extracellular vesicle ranges from 20nm to 1000nm, and may be contained in the internal space, displayed on the outer surface of the extracellular vesicle and / or across the membrane. The payload may include nucleic acids, proteins, carbohydrates, lipids, small molecules and / or combinations thereof. As an example and not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect operation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicle organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosomes fusion with plasma membranes). Extracellular vesicles may be derived from living or dead organisms, transplanted tissues or organs, and / or cultured cells.

[0073] As used herein, the term "exosome" refers to a cell-derived small (diameter between 20-300nm, more preferably between 40-200nm) vesicle, which includes a membrane that closes the internal space, and is produced by the cell through direct plasma membrane budding or by late endosome fusion with the plasma membrane. Exosomes are a type of extracellular vesicle. Exosomes contain lipids or fatty acids and polypeptides, and optionally contain a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a monosaccharide, a polysaccharide, or a polysaccharide) or other molecules. Exosomes can be derived from production cells and separated from production cells based on their size, density, biochemical parameters, or a combination thereof.

[0074] As used herein, the term "nanovesicle" refers to a cell-derived small (diameter between 20-250nm, more preferably between 30-150nm) vesicle, which includes a membrane that closes the internal space, and is produced by the cell by direct or indirect operation, so that the production cell will not produce the nanovesicle in the absence of the operation. Suitable operations of the production cell include, but are not limited to, continuous extrusion, treatment with an alkaline solution, ultrasonic treatment, or a combination thereof. In some cases, the production of nanovesicles may lead to the destruction of the production cell. Preferably, the population of nanovesicles is substantially free of vesicles derived from production cells by direct budding from the plasma membrane or late endosome fusion with the plasma membrane. The nanovesicle comprises lipids or fatty acids and polypeptides, and optionally comprises a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a monosaccharide, a polysaccharide, or a polysaccharide) or other molecules. Once the nanovesicle is derived from the production cell according to the operation, the nanovesicle can be isolated from the production cell based on its size, density, biochemical parameters, or a combination thereof.

[0075] As used herein, the term "luminal engineered exosome" refers to an exosome whose internal luminal space has been modified in composition. 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 previously modified by chemical, physical or biological methods. Specifically, the composition can be altered by genetic engineering, or by being produced by cells previously modified by genetic engineering.

[0076] As used herein, the term "modification" of a protein refers to a protein having at least 15% identity to the non-mutated amino acid sequence of the protein. Modifications of a protein include fragments or variants of the protein. Modifications of a protein may further include chemical or physical modifications to fragments or variants of the protein.

[0077] 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 a naturally occurring protein. Preferably, a fragment of MARCKS, MARCKSL1 or BASP1 retains the ability to specifically target the exosome cavity. Such a fragment is also referred to as a "functional fragment". Whether a fragment is a functional fragment in the sense described can be assessed by any method known in the art to determine the protein content of the exosome, including Western blot, FACS analysis, and fusion of a fragment with an autofluorescent protein such as GFP. In a specific embodiment, a fragment of MARCKS, MARCKSL1, or BASP1 retains at least 50%, 60%, 70%, 80%, 90% or 100% of the ability of naturally occurring MARCKS, MARCKSL1, or BASP1 to specifically target exosomes. In a specific 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 an exosome cavity, respectively. This ability can be assessed, for example, by fluorescently labeled variants in an assay as described in the experimental section.

[0078] As used herein, the term "variant" of a protein refers to a protein that shares a specific amino acid sequence identity with another protein after alignment by methods known in the art. Variants of a protein may include substitutions, insertions, deletions, frameshifts or rearrangements in another protein. In a specific embodiment, the variant is a variant with at least 70% identity to 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 with its functional fragment. 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 with its functional fragment. In some embodiments, the 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, preferably the variant or fragment variant retains the ability to specifically target an exosome lumen.

[0079] Methods of alignment of sequences 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). NCBI Basic Local Alignment Study Tool (BLAST) [Altschul et al., J. Mol. Biol. 215: 403-10 (1990)] can be obtained from several sources, including the National Center for Biological Information (NBCl, Bethesda, Md.) and on the Internet for use in conjunction with the sequence analysis programs blastp, blasm, blastx, tblastn and tblastx. BLAST and a description of how to use the program to determine sequence identity can be accessed on the official website of the NCBI (National Center for Biotechnology Information) under the NIH (National Institutes of Health).

[0080] The description 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 an exosome cavity. In a specific 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 an exosome cavity, respectively.

[0081] As used herein, the term "producer cell" refers to a cell used to produce exosomes. Producer cells can be cells cultured in vitro, or cells in vivo. Producer cells include, but are not limited to, cells known to effectively produce exosomes, such as HEK293 cells, Chinese hamster ovary (CHO) cells, and mesenchymal stem cells (MSCs).

[0082] As used herein, the term "target protein" or "target peptide" refers to a protein or peptide that can be targeted to an exosome cavity. The target protein or peptide can be a non-mutant protein that is naturally targeted to the exosome cavity, or a fragment or modification of the non-mutant protein. The target protein can be a fusion protein containing a flag tag, a therapeutic peptide, a targeting moiety, or other peptides attached to the non-mutant protein, or a modification or fragment of the non-mutant protein. The target protein can include modifications such as myristoylation, isoprenylation or palmitoylation, or a soluble protein attached to the inner leaflet of the membrane via a linker.

[0083] As used herein, the term "cargo protein" or "cargo peptide" refers to any protein or peptide, or a fragment or modification thereof, that can be loaded into an exosome or engineered exosome. The cargo protein or peptide may include a therapeutic peptide or protein that acts on a target (e.g., a target cell) in contact with the exosome. The cargo protein may be a fusion protein comprising a target protein or peptide as described above, or a fragment or modification thereof, such that the cargo fusion protein can be targeted to the exosome cavity.

[0084] 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 enclosed in an exosome and are not attached or bound to an exosome membrane.

[0085] As used herein, the terms "isolate," "isolated," and "isolating," or "purify," "purified," and "purifying," and "extracted," and "extracting," are used interchangeably and refer to the state of a preparation (e.g., a plurality of 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, partially removing (e.g., a portion) exosomes from a sample containing producer cells. In some embodiments, the isolated exosome composition has no detectable undesirable activity, or alternatively, the level or amount of undesirable activity is equal to or below an acceptable level or amount. In other embodiments, the isolated exosome composition has an amount and / or concentration of desired exosomes that is equal to or above an acceptable amount and / or concentration. In other embodiments, the isolated exosome composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition is obtained. Compared to the starting material, such enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.999%, 99.999%, or greater than 99.9999%. In some embodiments, the isolated exosome preparation is substantially free of residual biological products. In some embodiments, the isolated exosome preparation is 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free, 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 does not contain detectable producer cells, and only exosomes are detectable.

[0086] 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" covers any agent approved by a U.S. federal government regulatory agency or listed in the U.S. 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 eliminate the biological activity and properties of the administered compound. Excipients and carriers are included, which can be used to prepare pharmaceutical compositions and are generally safe, non-toxic and desirable.

[0087] As used herein, the term "payload" refers to a therapeutic agent that acts on a target (e.g., a target cell) that contacts an EV. Payloads that can be introduced into exosomes and / or production cells include therapeutic agents such as nucleotides (e.g., nucleotides that contain a detectable moiety or a toxin or disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes, or RNA molecules with regulatory functions such as miRNA, dsDNA, lncRNA, and siRNA), amino acids (e.g., amino acids that contain a detectable moiety or a toxin or disrupt 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).

[0088] As used herein, "mammalian subjects" include 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.).

[0089] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for 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.

[0090] As used herein, the term "substantially free" means that the sample containing exosomes contains less than 10% macromolecules in a mass / volume (m / v) percentage concentration. 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) macromolecules.

[0091] As used herein, the term "macromolecule" means nucleic acid, exogenous protein, lipid, carbohydrate, metabolite or a combination thereof.

[0092] As used herein, the term "conventional exosomal proteins" means proteins previously known to be enriched in exosomes, including but not limited to CD9, CD63, CD81, PDGFR, GPI anchors, lactadherins LAMP2 and LAMP2B, fragments thereof, or peptides that bind thereto. For the avoidance of doubt, PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporters, or fragments or variants thereof are not conventional exosomal proteins.

[0093] Other interpretation conventions

[0094] The ranges listed herein are to be understood as a shorthand for all values ​​within the range (including the listed endpoints). For example, a range of 1 to 50 is to be understood to include any value, combination of values, or subrange 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.

[0095] Unless otherwise indicated, reference to compounds having one or more stereocenters refers to each stereoisomer and all combinations of stereoisomers thereof.

[0096] Exosome Protein

[0097] Some embodiments of the present invention relate to the identification, use and modification of exosomal proteins that are highly enriched in the lumen of exosomes. Such exosomal proteins can be identified by analyzing highly purified exosomes using mass spectrometry or other methods known in the art.

[0098] The proteins include various luminal proteins or membrane proteins enriched in the exosome membrane, such as transmembrane proteins, integral proteins 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).

[0099] Depending on the production cell, production conditions, purification method, or intended application of the exosomes, one or more exosomal proteins identified herein may be selectively used. Exosomal proteins enriched in certain exosome cavities having a specific size range, targeting moiety, charge density, payload, etc. may be identified and used in some embodiments of the present invention. In some embodiments, more than one exosomal protein may be used simultaneously or subsequently to produce and isolate therapeutic exosomes.

[0100] Luminal engineered exosomes

[0101] Another aspect of the invention relates to the production and use of lumen-engineered exosomes. Lumen-engineered exosomes have an interior space whose composition is modified. For example, the composition of the lumen can be modified by changing the protein, lipid or glycan content of the lumen.

[0102] In some embodiments, lumen-engineered exosomes are produced by chemical and / or physical methods (such as PEG-induced fusion and / or ultrasound fusion).

[0103] In other embodiments, the lumen-engineered exosomes are produced by genetic engineering. Exosomes produced by genetically modified production cells or the offspring of genetically modified cells can contain modified lumen compositions. In some embodiments, lumen-engineered exosomes have a higher or lower density of exosomal proteins, or include modifications or fragments of exosomal proteins.

[0104] For example, lumen-engineered exosomes can be produced by cells transformed with exogenous sequences encoding exosomal proteins or exosomal protein modifications or fragments. Exosomes comprising proteins expressed by exogenous sequences can include modified lumenal protein compositions.

[0105] 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 cavity can be used. Proteins modified to contain the minimum fragment required for specific and effective targeting to the exosome cavity can also be used.

[0106] Fusion proteins with 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.

[0107] Therapeutic peptides are selected from the group consisting of natural peptides, recombinant peptides, synthetic peptides or joints to therapeutic compounds. Therapeutic compounds can be nucleotides, amino acids, lipids, carbohydrates or small molecules. Therapeutic peptides can be antibodies, enzymes, ligands, antigens (e.g., tumor antigens or antigens from infectious agents such as bacteria, viruses, fungi or protozoa), receptors, antimicrobial peptides, transcription factors or their fragments or modifiers. Fusion proteins can be presented in exosome cavities and provide therapeutic activity for exosomes.

[0108] 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.

[0109] In some embodiments, the therapeutic peptide is a transmembrane peptide. The transmembrane peptide 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 the cavity sequence in the exosome cavity, and a second end expressed on the exosome surface. In some embodiments, the transmembrane protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment or variant thereof.

[0110] 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, MS2 phage 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.

[0111] In some embodiments, the therapeutic peptide is part of a protein-protein interaction system. In some embodiments, the protein-protein interaction system includes a FRB-FKBP interaction system, for example, a FRB-FKBP interaction system as described in Banaszynski et al., J Am Chem Soc. 2005 Apr 6; 127(13):4715-21.

[0112] The fusion protein can be targeted to the exosome lumen and provide therapeutic activity to the exosome.

[0113] In some embodiments, a fusion protein with a targeting portion is used. For example, the fusion protein can include MARCKS, MARCKSL1, BASP1, or a fragment or modifier thereof, and a targeting portion. The targeting portion can be used to target exosomes to specific organs, tissues or cells to treat using exosomes. In some embodiments, the targeting portion is an antibody or its antigen-binding fragment. Antibodies and their antigen-binding fragments include whole antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, fragments thereof, and further include single-chain antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, mouse-human antibodies, mouse-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, double antibodies and antibody-related polypeptides. Antibodies and their antigen-binding fragments also include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0114] In some embodiments, fusion proteins comprising viral proteins are used. In some embodiments, the fusion proteins comprise viral capsid or envelope proteins. In some embodiments, the fusion proteins allow assembly of intact viruses that are retained in the exosome cavity.

[0115] In some embodiments, a fusion protein comprising MARCKS, MARCKSL1, BASP1, any one of SEQ ID NOs: 4-109, or a modification thereof, in particular a fragment or variant thereof, results in enrichment of the fusion protein in exosomes compared to a fusion protein lacking MARCKS, MARCKSL1, BASP1, any one of SEQ ID NOs: 4-109, or a modification thereof, in particular a fragment or variant thereof, or compared to a fusion protein comprising a conventional exosomal protein. In some embodiments, a fusion protein comprising MARCKS, MARCKSL1, BASP1, any one of SEQ ID NOs: 4-109, or a fragment or modification thereof, in particular a fragment or variant thereof, comprises a peptide having the sequence MGXKLSKKK, wherein 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)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position 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)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is ​​neither (+) nor (Asp or Glu). In some embodiments, conventional exosomal proteins are selected from the list consisting of CD9, CD63, CD81, PDGFR, GPI anchor protein, LAMP2, LAMP2B, and fragments thereof.In some embodiments, the enrichment of fusion proteins comprising MARCKS, MARCKSL1, BASP1, any one of SEQ ID NOs: 4-109, or fragments or modifications 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 fusion proteins lacking MARCKS, MARCKSL1, BASP1, any one of SEQ ID NOs: 4-109, or fragments or modifications thereof, or fusion proteins comprising conventional exosomal proteins. In some embodiments, the protein sequence of any one of SEQ ID NOs: 1-109 is sufficient to load exosomes with the fusion protein.

[0116] In some embodiments, the lumen-engineered exosomes comprising fusion proteins containing exogenous sequences and exosomal lumenal proteins newly identified herein have a higher fusion protein density than similarly engineered exosomes containing exogenous sequences conjugated to conventional exosomal proteins known in the art (e.g., CD9, CD63, CD81, PDGFR, GPI anchors, lactadherins LAMP2 and LAMP2B, fragments thereof, or peptides bound thereto). In some embodiments, the fusion proteins containing the exosomal proteins newly identified herein are present in the exosomal lumen at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to fusion proteins in other exosomal lumens similarly modified with conventional exosomal proteins. In some embodiments, the fusion protein containing the exosomal protein newly identified herein is present in the exosome lumen at a density of 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times or more compared to fusion proteins in other exosome lumens similarly modified with conventional exosome proteins.

[0117] In some embodiments, compared to exosomes similarly modified with CD9, fusion proteins comprising MARCKS, variants, fragments, fragment variants, or modifications thereof are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more. In some embodiments, compared to exosomes similarly modified with CD63, fusion proteins comprising MARCKS, variants, fragments, fragment variants, or modifications thereof are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more. In some embodiments, compared to exosomes similarly modified with CD81, fusion proteins comprising MARCKS, variants, fragments, fragment variants, or modifications thereof are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more. In some embodiments, compared to exosomes similarly modified with PDGFR, fusion proteins comprising MARCKS, its variants, fragments, fragment variants, or modifications are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more. In some embodiments, compared to exosomes similarly modified with GPI anchor proteins, fusion proteins comprising MARCKS, its variants, fragments, fragment variants, or modifications are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more. In some embodiments, compared to exosomes similarly modified with lactadherin, fusion proteins comprising MARCKS, its variants, fragments, fragment variants, or modifications are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more. In some embodiments, compared to exosomes similarly modified with LAMP2, fusion proteins comprising MARCKS, its variants, fragments, fragment variants, or modifications are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more. In some embodiments, compared to exosomes similarly modified with LAMP2B, fusion proteins comprising MARCKS, its variants, fragments, fragment variants, or modifications are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more. In some embodiments, compared to exosomes similarly modified with conventional proteins, fusion proteins comprising MARCKS, its variants, fragments, fragment variants, or modifications are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more.In some embodiments, fusion proteins comprising MARCKS, variants, fragments, fragment variants, or modifications thereof are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified using variants of conventional exosomal proteins.

[0118] In some embodiments, the fusion protein comprising MARCKSL1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with CD9. In some embodiments, the fusion protein comprising MARCKSL1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with CD63. In some embodiments, the fusion protein comprising MARCKSL1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with CD81. In some embodiments, the fusion protein comprising MARCKSL1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with PDGFR. In some embodiments, the fusion protein comprising MARCKSL1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with GPI anchors. In some embodiments, the fusion protein comprising MARCKSL1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with lactadherin. In some embodiments, the fusion protein comprising MARCKSL1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with LAMP2. In some embodiments, the fusion protein comprising MARCKSL1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with LAMP2B. In some embodiments, the fusion protein comprising MARCKSL1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with conventional proteins.In some embodiments, fusion proteins comprising MARCKSL1, a variant, fragment, fragment variant, or modification thereof are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified using variants of conventional exosomal proteins.

[0119] In some embodiments, the fusion protein comprising BASP1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with CD9. In some embodiments, the fusion protein comprising BASP1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with CD63. In some embodiments, the fusion protein comprising BASP1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with CD81. In some embodiments, the fusion protein comprising BASP1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with PDGFR. In some embodiments, the fusion protein comprising BASP1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with GPI anchors. In some embodiments, the fusion protein comprising BASP1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with lactadherin. In some embodiments, the fusion protein comprising BASP1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with LAMP2. In some embodiments, the fusion protein comprising BASP1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with LAMP2B. In some embodiments, the fusion protein comprising BASP1, its variant, fragment, fragment variant, or modification is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with conventional proteins.In some embodiments, fusion proteins comprising BASP1, variants, fragments, fragment variants, or modifications thereof are present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified using variants of conventional exosomal proteins.

[0120] In some embodiments, the fusion protein comprising any one of SEQ ID NOs: 1-109, variants, fragments, fragment variants, or modifications thereof is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with CD9. In some embodiments, the fusion protein comprising any one of SEQ ID NOs: 1-109, variants, fragments, fragment variants, or modifications thereof is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with CD63. In some embodiments, the fusion protein comprising any one of SEQ ID NOs: 1-109, variants, fragments, fragment variants, or modifications thereof is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with CD81. In some embodiments, the fusion protein comprising any one of SEQ ID NOs: 1-109, variants, fragments, fragment variants, or modifications thereof is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with PDGFR. In some embodiments, a fusion protein comprising any one of SEQ ID NOs: 1-109, a variant, fragment, fragment variant, or modification thereof is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with a GPI anchor. In some embodiments, a fusion protein comprising any one of SEQ ID NOs: 1-109, a variant, fragment, fragment variant, or modification thereof is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with a lactadherin. In some embodiments, the fusion protein comprising any one of SEQ ID NOs: 1-109, variants, fragments, fragment variants, or modifications thereof is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with LAMP2. In some embodiments, the fusion protein comprising any one of SEQ ID NOs: 1-109, variants, fragments, fragment variants, or modifications thereof is present at a density 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with LAMP2B.In some embodiments, a fusion protein comprising any one of SEQ ID NOs: 1-109, a variant, fragment, fragment variant, or modification thereof is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with conventional proteins. In some embodiments, a fusion protein comprising any one of SEQ ID NOs: 1-109, a variant, fragment, fragment variant, or modification thereof is present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more compared to exosomes similarly modified with variants of conventional exosomal proteins.

[0121] In some embodiments, compared with the cavity engineering exosomes known in the art, the cavity engineering exosomes described herein show excellent characteristics. For example, the cavity engineering exosomes produced by using the newly identified exosome proteins provided herein are compared with the exosomes in the prior art (such as those produced using conventional exosome proteins), and contain a more highly enriched modified protein in its cavity. In addition, compared with the cavity engineering exosomes known in the art, the cavity engineering exosomes of the present invention can have larger, more specific or more controllable biological activity. For example, the cavity engineering exosomes comprising therapeutic or biologically related exogenous sequences fused with exosome proteins or fragments thereof described herein (such as BASP1 or fragments thereof) can have more desired engineering features than fusion with scaffolds known in the art. Scaffold proteins known in the art include tetraspanins (e.g., CD63, CD81, CD9, etc.), lysosomal associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI anchors, lactadherins and fragments thereof, and peptides having 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 random or random distribution of exogenous proteins in exosomes to produce cavity-engineered exosomes. This resulted in low levels of exogenous proteins in exosomes, unpredictable density. Therefore, the exosome proteins and fragments thereof described herein provide important advances in new exosome compositions and methods for preparing them.

[0122] The fusion proteins provided herein may comprise MARCKS, MARCKSL1, BASP1 or a fragment or variant thereof and an additional peptide. The additional peptide may be attached to the N-terminus or C-terminus of the exosomal protein or a fragment or variant thereof.

[0123] In some embodiments, the fusion proteins provided herein comprise MARCKS, MARCKSL1, BASP1, or a fragment or variant thereof and two additional peptides. The two additional peptides can be attached to the N-terminus or C-terminus of the 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.

[0124] In some embodiments, the compositions and methods described herein for producing lumen-engineered extracellular vesicles comprise nanovesicles.

[0125] Producer cells for the production of lumen-engineered exosomes

[0126] The exosomes of the present invention can be produced by cells grown in vitro or body fluids of a subject. When exosomes are produced by in vitro cell culture, various production cells, such as HEK293 cells, can be used in the present invention. Other cell types that can be used to produce the cavity engineered exosomes described herein include, but are not limited to, mesenchymal stem cells, T cells, B cells, dendritic cells, macrophages, and cancer cell lines.

[0127] The production cell can be genetically modified to contain one or more exogenous sequences to produce cavity engineered exosomes. The genetically modified production cell can contain exogenous sequences transformed transiently or stably. The exogenous sequence can be transformed as a plasmid. The exogenous sequence can be stably integrated into the genomic sequence of the production cell at a target site or a random site. In some embodiments, a stable cell line is generated for the production of cavity engineered exosomes.

[0128] The exogenous sequence can be inserted into the genomic sequence of the production cell, located 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 sequence into the production cell. For example, cells modified using various gene editing methods (e.g., using homologous recombination, transposon-mediated systems, loxP-Cre systems, CRISPR / Cas9 or TALEN methods) are within the scope of the present invention.

[0129] The exogenous sequence may include a sequence encoding an exosomal protein or a modification or fragment of the exosomal protein. Additional copies of sequences encoding exosomal proteins may be introduced to produce cavity-engineered exosomes with a higher density of exosomal proteins. Exogenous sequences encoding modifications or fragments of exosomal proteins may be introduced to produce cavity-engineered exosomes containing modifications or fragments of exosomal proteins. Exogenous sequences encoding affinity tags may be introduced to produce cavity-engineered exosomes containing fusion proteins comprising affinity tags attached to exosomal proteins.

[0130] In some embodiments, compared with the natural exosomes separated from the same or similar production cell type, the cavity engineering exosome has a higher density of exosome proteins. In some embodiments, compared with the natural exosomes, the exosome proteins are present at a density of 2, 4, 8, 16, 32, 64, 100, 200, 400, 800, 1,000 times or more on the cavity engineering exosomes. In some embodiments, compared with the natural exosomes, the exosome proteins are present at a density of 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times or more on the cavity engineering exosomes. In some embodiments, the fusion protein comprising the exosomal protein is present on the cavity engineered exosome at a density of 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times or more compared to the unmodified exosomal protein on the natural exosome. In some embodiments, the fragment or variant of the exosomal protein is present on the cavity engineered exosome at a density of 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times or more compared to the unmodified exosomal protein on the natural exosome.

[0131] In specific embodiments, MARCKS, a fragment or variant of MARCKS, or a modification thereof, is present at a density of 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times or more on the lumen-engineered exosomes compared to unmodified MARCKS on the native exosomes. In some embodiments, MARCKSL1, a fragment or variant of MARCKSL1, or a modification thereof is present at a density of 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times or more on the lumen-engineered exosomes compared to unmodified MARCKSL1 on the native exosomes. In some embodiments, BASP1, a fragment or variant of BASP1, or a modification thereof is present at a density of 2 to 4 times, 4 to 8 times, 8 to 16 times, 16 to 32 times, 32 to 64 times, 64 to 100 times, 100 to 200 times, 200 to 400 times, 400 to 800 times, 800 to 1,000 times, or more on the lumen-engineered exosomes compared to unmodified BASP1 on the native exosomes.

[0132] In some embodiments, the production cell is further modified to include additional exogenous sequences. For example, additional exogenous sequences can be included to regulate endogenous gene expression, or to produce exosomes containing a specific polypeptide as a payload. In some embodiments, the production cell is modified to include two exogenous sequences, one encoding an exosome protein or a modification or fragment of the exosome protein, and the other encoding a payload.

[0133] More specifically, lumen-engineered exosomes can be produced by cells transformed with sequences encoding one or more exosomal lumenal 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 exosomal lumenal proteins described herein can be expressed from a plasmid, an exogenous sequence inserted into the genome, or other exogenous nucleic acid such as a synthetic messenger RNA (mRNA).

[0134] In some embodiments, the one or more exosome coelomin proteins are expressed in cells transformed with an exogenous sequence encoding their full-length endogenous forms. In some embodiments, the exogenous sequence encodes the MARCKS protein of SEQ ID NO: 1. In some embodiments, the exogenous sequence encodes the MARCKSL1 protein of SEQ ID NO: 2. In some embodiments, the exogenous sequence encodes the BASP1 protein of SEQ ID NO: 3.

[0135] Luminally engineered exosomes can be produced by cells transformed with sequences encoding one or more exosomal lumenal protein fragments, 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 lumenal protein fragment that lacks 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 lumenal protein fragment that lacks 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 lumenal protein fragment that lacks at least 5, 10, 50, 100, 200, or 300 amino acids from the N-terminus and C-terminus of the native protein. In some embodiments, the sequence encodes an exosome coelomin fragment lacking one or more functional or structural domains of the native protein. In some embodiments, the fusion protein comprises a peptide of SEQ ID NO: 4-109. In some embodiments, the fusion protein comprises a peptide of SEQ ID NO: 13. In some embodiments, the fusion protein comprises a peptide having the sequence MGXKLSKKK, wherein 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)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position 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)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is ​​neither (+) nor (Asp or Glu).

[0136] In some embodiments, the cavity engineered exosome can be produced by cells transformed with sequences encoding exosome proteins fused to one or more heterologous proteins or their fragments or modifiers. In some embodiments, one or more heterologous proteins are fused to the N-terminus of an exosome protein or its modifier, particularly its fragment or variant. In some embodiments, one or more heterologous proteins are fused to the C-terminus of an exosome protein or its modifier, particularly its fragment or variant. In some embodiments, one or more heterologous proteins are fused to the N-terminus and C-terminus of an exosome protein or its modifier, particularly its fragment or variant. In some embodiments, the one or more heterologous proteins are fused to the N-terminus and C-terminus of an exosome protein or its modifier, particularly its fragment or variant. In some embodiments, the one or more heterologous proteins are mammalian proteins. In some embodiments, the one or more heterologous proteins are human proteins.

[0137] In some embodiments, the lumen-engineered exosomes are produced by cells transformed with a sequence encoding a polypeptide having a sequence identical or similar to the full length or fragment of a natural exosome lumen protein, 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 polypeptide is 50% identical to the full length or fragment of a natural exosome lumen protein, such as 50% identical to SEQ ID NO: 1-3. In some embodiments, the polypeptide is 60% identical to the full length or fragment of a natural exosome lumen protein, such as 60% identical to SEQ ID NO: 1-3. In some embodiments, the polypeptide is 70% identical to the full length or fragment of a natural exosome lumen protein, such as 70% identical to SEQ ID NO: 1-3. In some embodiments, the polypeptide is 80% identical to the full length or fragment of a native exosome coelomin, for example, 80% identical to SEQ ID NO: 1-3. In some embodiments, the polypeptide is 90% identical to the full length or fragment of a native exosome coelomin, for example, 90% identical to SEQ ID NO: 1-3. In some embodiments, the polypeptide is 95% identical to the full length or fragment of a native exosome coelomin, for example, 95% identical to SEQ ID NO: 1-3. In some embodiments, the polypeptide is 99% identical to the full length or fragment of a native exosome coelomin, for example, 99% identical to SEQ ID NO: 1-3. In some embodiments, the polypeptide is 99.9% identical to the full length or fragment of a native exosome coelomin, for example, 99.9% identical to SEQ ID NO: 1-3.

[0138] In some embodiments, the lumen-engineered exosomes produced by the 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 fragment of BASP1, for example, 50% identical to SEQ ID NO: 4-109. In some embodiments, the polypeptide is 60% identical to the full length or fragment of BASP1, for example, 60% identical to SEQ ID NO: 4-109. In some embodiments, the polypeptide is 70% identical to the full length or fragment of BASP1, for example, 70% identical to SEQ ID NO: 4-109. In some embodiments, the polypeptide is 80% identical to the full length or fragment of BASP1, for example, 80% identical to SEQ ID NO: 4-109. In some embodiments, the polypeptide is 90% identical to the full length or fragment of BASP1, for example, 90% identical to SEQ ID NO: 4-109. In some embodiments, the polypeptide is 95% identical to the full length or fragment of BASP1, for example, 95% identical to SEQ ID NO: 4-109. In some embodiments, the polypeptide is 99% identical to the full length or fragment of BASP1, for example, 99% identical to SEQ ID NO: 4-109. In some embodiments, the polypeptide is 99.9% identical to the full length or fragment of BASP1, for example, 99.9% identical to SEQ ID NO: 4-109. In some embodiments, the polypeptide is 100% identical to a fragment of BASP1, for example, 100% identical to SEQ ID NO: 4-109.

[0139] Characterization of exosomes

[0140] In some embodiments, the methods described herein further include 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.

[0141] Measurement of exosome content

[0142] Exosomes can include proteins, peptides, RNA, DNA, and lipids. Total RNA can be extracted using an acid-phenol:chloroform extraction method. Then, RNA can be purified using a glass fiber filter under conditions that recover total RNA containing small RNAs, or under conditions that separate small RNA species less than 200 nucleotides in length from longer RNA species such as mRNA. Because RNA is eluted in a small volume, an alcohol precipitation step may not be required to separate RNA.

[0143] Exome composition can be assessed by methods known in the art including, but not limited to, transcriptomics, sequencing, proteomics, mass spectrometry, or HP-LC.

[0144] Nucleotide composition (including RNA and DNA) associated with isolated exosomes can be measured using a variety of techniques well known to those skilled in the art (e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis, solution hybridization detection). In a specific embodiment, the level of at least one RNA is measured by reverse transcribing RNA from an 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 comprising RNA-specific probe oligonucleotides) to provide a hybridization spectrum of the exosome composition, and comparing the hybridization spectrum of the exosome composition with the hybridization spectrum produced by the control sample. The change in the signal of at least one RNA in the test sample relative to the control sample indicates the composition of the RNA.

[0145] In addition, the gene-specific oligonucleotide probes produced by known RNA sequences can be used to prepare microarrays. The array can contain two different oligonucleotide probes for every kind of RNA, one containing active mature sequences, and the other has specificity to the precursors of RNA (e.g., miRNA and pre-miRNA). The array can also contain controls, such as one or more mouse sequences that only differ by a few bases from human orthologs, which can be used as controls for hybridization stringency conditions. TRNA and other RNAs (e.g., rRNA, mRNA) from two species can also be printed on the microchip, thereby providing internal, relatively stable positive controls for specific hybridization. One or more suitable controls for nonspecific hybridization can also be included on the microchip. For this reason, select sequences based on having no homology with any known RNA.

[0146] Microarrays can be made 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 are prepared using a commercially available microarray system (e.g., GeneMachine OmniGrid. TM. 100 microarrays and Amersham CodeLink. TM) is printed on an activated slide. A labeled cDNA oligomer corresponding to the target RNA is prepared by reverse transcribing the target RNA with a labeled primer. After the first strand is synthesized, the RNA / DNA hybrid is denatured to degrade the RNA template. The labeled target cDNA thus prepared is then hybridized to a microarray chip under hybridization conditions, such as 6 times. SSPE / 30% formamide lasts for 18 hours at 25°C, followed by 0.75. times washing. TNT lasts for 40 minutes at 37°C. Hybridization occurs at the position of the complementary target cDNA in the sample where the probe DNA is fixed on the array. The labeled target cDNA marks the exact position on the array where binding occurs, allowing automatic detection and quantification. The output consists of a series of hybridization events, thereby indicating the relative abundance of a specific cDNA sequence in an exosome preparation, and therefore the relative abundance of the corresponding complementary RNA. According to one embodiment, the labeled cDNA oligomer is a biotin-labeled cDNA, prepared by a biotin-labeled primer. The microarray is then processed by direct detection of biotin-containing transcripts using, for example, streptavidin-Alexa647 conjugates 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.

[0147] Data mining is done by bioinformatics, including scanning chips, signal acquisition, image processing, normalization, statistical processing and data comparison and path analysis. Therefore, microarrays can analyze hundreds of polynucleotides simultaneously with high-throughput performance. Microarray spectrum analysis of mRNA expression has successfully provided valuable data for gene expression research 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. The analysis of 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.

[0148] Measurement of exosome size

[0149] In some embodiments, the methods described herein include measuring the size of exosomes and / or exosome populations contained in the purified fraction. In some embodiments, exosome size is measured as the longest measurable dimension. In general, the longest general dimension of an exosome is also referred to as its diameter.

[0150] Exosome size 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.

[0151] 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 assays (NTA, for example, using a Malvern Nanosight NS300 nanoparticle tracking device). In a specific embodiment, exosome size is determined using Malvern NanoSight NS300. In some embodiments, exosomes described herein have a longest dimension of about 20-1000nm as measured by NTA (for example, using a Malvern Nanosight NS300). In other embodiments, exosomes described herein have a longest dimension of about 40-1000nm as measured by NTA (for example, using a Malvern Nanosight NS300). In other embodiments, exosome populations described herein include populations, wherein 90% of the exosomes have a longest dimension of about 20-1000nm as measured by NTA (for example, using a Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population, wherein 95% of the exosomes have a longest dimension of about 20-1000nm as measured by NTA (e.g., using Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population, wherein 99% of the exosomes have a longest dimension of about 20-1000nm as measured by NTA (e.g., using Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population, wherein 90% of the exosomes have a longest dimension of about 40-1000nm as measured by NTA (e.g., using Malvern Nanosight NS300). In other embodiments, the exosome population described herein includes a population, wherein 95% of the exosomes have a longest dimension of about 40-1000nm as measured by NTA (e.g., using Malvern Nanosight NS300). In other embodiments, a population of exosomes described herein comprises a population wherein 99% of said exosomes have a longest dimension of about 40-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300).

[0152] Exosome size can be measured using adjustable resistive pulse sensing (TRPS). In a specific embodiment, the size of the exosome as measured by TRPS is determined using iZON qNANO Gold. In some embodiments, the exosomes described herein have a longest dimension of about 20-1000nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the exosomes described herein have a longest dimension of about 40-1000nm as measured by TRPS (e.g., iZON qNano Gold). In other embodiments, the exosome populations described herein include a population, wherein 90% of the exosomes have a longest dimension of about 20-1000nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the exosome populations described herein include a population, wherein 95% of the exosomes have a longest dimension of about 20-1000nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the exosome populations described herein include a population, wherein 99% of the exosomes have a longest dimension of about 20-1000nm as measured by TRPS (e.g., using iZON qNanoGold). In other embodiments, the exosome populations described herein include a population, wherein 90% of the exosomes have a longest dimension of about 40-1000nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the exosome populations described herein include a population, wherein 95% of the exosomes have a longest dimension of about 40-1000nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the exosome populations described herein include a population, wherein 99% of the exosomes have a longest dimension of about 40-1000nm as measured by TRPS (e.g., using iZON qNano Gold).

[0153] Exosome size can be measured using electron microscopy. In some embodiments, the electron microscopy method used to measure exosome size is transmission electron microscopy. In a specific embodiment, the transmission electron microscope used to measure exosome size is a Tecnai TM G2Spirit BioTWIN. Methods for measuring exosome size using electron microscopy are well known to those skilled in the art, and any such method may be adapted to measure exosome size. In some embodiments, the exosomes described herein have a size as determined by scanning electron microscopy (e.g., Tecnai TMG2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosomes described herein have a longest dimension of about 20-1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome population described herein includes a population wherein 90% of the exosomes have a maximum dimension of about 40-1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome population described herein includes a population wherein 95% of the exosomes have a maximum dimension of about 20-1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM G2Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome population described herein includes a population wherein 99% of the exosomes have a maximum dimension of about 20-1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome population described herein includes a population wherein 90% of the exosomes have a maximum dimension of about 20-1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM G2 SpiritBioTWIN scanning electron microscope). In other embodiments, the exosome population described herein includes a population wherein 95% of the exosomes have a maximum dimension of about 40-1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM G2Spirit BioTWIN scanning electron microscope). In other embodiments, the exosome population described herein includes a population wherein 99% of the exosomes have a maximum dimension of about 40-1000 nm as measured by a scanning electron microscope (e.g., Tecnai TM The longest dimension was approximately 40-1000 nm as measured by G2 Spirit BioTWIN scanning electron microscope.

[0154] Individual exosome size can be determined based on particles one by one by nano flow cytometry. In some embodiments, nano flow cytometer is a flow nano analyzer (NanoFCM company; Xiamen, China). In some embodiments, exosomes described herein have the longest dimension of about 20-1000nm as measured by nano flow cytometry (e.g., using a flow nano analyzer). In some embodiments, exosomes described herein have the longest dimension of about 40-1000nm as measured by nano flow cytometry (e.g., using a flow nano analyzer). In some embodiments, exosome colonies described herein include colonies, wherein 90% of the exosomes have the longest dimension of about 20-1000nm as measured by nano flow cytometry (e.g., using a flow nano analyzer). In some embodiments, exosome colonies described herein include colonies, wherein 95% of the exosomes have the longest dimension of about 20-1000nm as measured by nano flow cytometry (e.g., using a flow nano analyzer). In some embodiments, the exosome population described herein includes a colony, wherein 99% of the exosomes have a longest dimension of about 20-1000nm as measured by nano flow cytometry (e.g., using a flow nano analyzer). In some embodiments, the exosome population described herein includes a colony, wherein 90% of the exosomes have a longest dimension of about 40-1000nm as measured by nano flow cytometry (e.g., using a flow nano analyzer). In some embodiments, the exosome population described herein includes a colony, wherein 95% of the exosomes have a longest dimension of about 40-1000nm as measured by nano flow cytometry (e.g., using a flow nano analyzer). In some embodiments, the exosome population described herein includes a colony, wherein 99% of the exosomes have a longest dimension of about 40-1000nm as measured by nano flow cytometry (e.g., using a flow nano analyzer).

[0155] Measurement of exosome charge density

[0156] In some embodiments, the methods described herein comprise measuring the charge density of exosomes and / or exosome populations contained in the purified fractions. In some embodiments, the charge density is measured by potentiometric titration, anion exchange, cation exchange, isoelectric focusing, zeta potential, capillary electrophoresis, capillary zone electrophoresis, or gel electrophoresis.

[0157] Measurement of exosome protein density

[0158] 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, protein quantity 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, for example, by using biolayer interferometry (BLI), FACS, Western blotting, fluorescence (e.g., GFP-fusion protein) detection, nano-flow cytometry, ELISA, αLISA and / or by measuring the bands on the protein gel for density measurement.

[0159] Example

[0160] The following examples are presented in order to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of the inventors' invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Although every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, for example, bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; etc.

[0161] Unless otherwise indicated, the practice of the present invention will employ conventional protein chemistry, biochemistry, recombinant DNA technology and pharmaceutical methods within the art. Such techniques are fully described in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL. 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, ed., 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).

[0162] Example 1: Identification of novel exosomal proteins

[0163] Collection of exosomes

[0164] Exosomes were collected from the supernatant of high-density suspension cultures of HEK293 SF cells after 9 days. 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 separated and eluted in Optiprep TM Further fractionation was performed by ultracentrifugation on a density gradient.

[0165] For Optiprep TM Gradient, in a 12 mL Ultra-Clear (344059) tube in a SW 41Ti rotor, with 4 mL 45% Optiprep TM 、3mL 30%Optiprep TM 、2mL22.5%Optiprep TM , 2mL 17.5%Optiprep TM Prepare a 4-layer sterile gradient with 1 mL of PBS. Add the exosome fraction to the Optiprep TMThe exosome fraction was isolated by ultracentrifugation at 200,000 x g for 16 hours at 4°C in a gradient. Ultracentrifugation produced 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 at the top of the tube.

[0166] 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 pellet the purified exosomes. The pelleted exosomes were then resuspended in a minimal volume of PBS (approximately 200 μL) and stored at 4° C.

[0167] Sample preparation for LC-MS / MS analysis

[0168] To determine proteins specific to exosomes, Optiprep TM The top fraction and bottom fraction of the gradient. Before analysis, the total protein concentration of the two samples was determined by bicinchoninic acid (BCA) assay, and each sample was appropriately diluted to 125 μg / mL in PBS buffer. Next, 50.0 μL of each sample was added to a separate 1.5 mL microcentrifuge tube 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 transfer of 2.0 μL 1.0% Triton X-100 solution. All samples were then incubated at 55 ° C for 60 minutes.

[0169] Protein precipitation is carried out by adding 1250 μ L ethanol at -20 ℃. In order to improve efficiency, the sample is vortexed violently and then ultrasonicated in a water bath for 5 minutes. The material of precipitation is precipitated by centrifugation at room temperature with 15,000g for 5 minutes. The supernatant is decanted, and the material of precipitation is thoroughly dried using nitrogen. The precipitation is resuspended in 30.0 μ L digestion buffer (30 mMTris, 1.0 M GdmCl, 100 mM EDTA, 50 mM TCEP, pH 8.5), which also reduces disulfide bonds. Alkylate free cysteine ​​residues by adding 5.0 μ L 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.

[0170] After incubation, 30.0 μ L 50 mM Tris pH 8.5 was used to dilute each sample, and proteolytic digestion was started by adding 2.0 μ g trypsin. All samples were mixed, and then incubated overnight at 37 ° C. After incubation, trypsin activity was stopped by adding 5.0 μ L 10% formic acid. Before analysis by LC-MS / MS, each sample was desalted using a Pierce C18 spin column. At the end of the process, each sample was dried and reconstituted with 0.1% formic acid in 75.0 μ L of 95:5 water: acetonitrile, and transferred to an HPLC bottle for analysis.

[0171] LC-MS / MS analysis

[0172] The samples were injected into an UltiMate 3000RSCLnano (Thermo Fisher Scientific) low flow chromatography system, and the tryptic peptides were loaded onto an Acclaim PepMap 100C18 trapping column (75 μm x 2 cm, 3 μm particle size, 10 μm x 20 μm) at a flow rate of 2.500 μL / min using a loading mobile phase (MPL: 95% water, 5% acetonitrile, 0.1% formic acid). The sample was passed through an EASY-Spray LC C18 analytical column (75 μm x 25 cm, 2 μm particle size, Thermo Fisher Scientific) at a flow rate of 300 nL / min. The peptides were eluted and separated using a gradient of mobile phase A (MPA: water, 0.1% formic acid) and mobile phase B (MPB: acetonitrile, 0.1% formic acid) using a 100 μM pore size, Thermo Fisher Scientific). The step gradient used for elution started with 5% MPB, which was maintained for 15 minutes during loading. The MPB percentage was then increased from 5% to 17% in 30 minutes, from 17% to 25% in 45 minutes, and finally from 25% to 40% in 5 minutes. The most hydrophobic materials were removed by increasing to 90% MPB in 5 minutes and then holding there for 9 minutes. The total run time of the method was 130 minutes, and sufficient time was allowed for column re-equilibration. Wash cycles were performed between analytical injections to minimize carryover.

[0173] Mass analysis was performed with a Q Exactive Basic (Thermo Fisher Scientific) mass spectrometer. Precursor ion mass spectra were measured at a resolution of 70,000 in the m / z range of 400-1600Da. The 10 strongest precursor ions were selected and fragmented in the HCD cell using a collision energy of 27, and the MS / MS spectra were measured at a resolution of 35,000 in the m / z range of 200-2000Da. Ions with charge states 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 the misidentification of known contaminants.

[0174] Data processing

[0175] Proteins were first 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. Searches were performed against the complete Uniprot Homo sapiens (classification 9606 version: 127,783 entries) or Swiss-Prot Homo sapiens (classification 9606 version 2017-05-10: 42,153 entries) reference databases, as well as a custom Uniprot database containing E1a proteins (7 entries). The following search parameters were used: enzyme, trypsin; maximum of 2 missed cleavages; minimum peptide length of 6 residues; 10 ppm precursor mass tolerance; and 0.02 Da fragment mass tolerance. The search also included specific dynamic modifications (oxidation of M; deamidation of N or Q; phosphorylation of S, T, or Y; pyroglutamination of peptide terminus E; and acetylation of protein N terminus) and static modifications (carbamidomethylation of C).

[0176] In the Target Decoy PSM validator, the maximum δCn and the stringent and relaxed target false discovery rates (FDR) were all set to 1 as the data were searched again using Scaffold software (version 4.8.2, Proteome Software Inc.). In Scaffold, the data were also searched using the X! Tandem open source algorithm to identify proteins using a 99.0% protein threshold, a minimum of 2 peptides, and a 95% peptide threshold.

[0177] To determine the identity of novel exosome-specific proteins, Optiprep TM The exosome fraction at the top of the gradient was compared for total peptide spectrum matches (PSM) against those found in the lower fractions. Figure 2As shown, there is a weak correlation between the top fraction proteins (Y-axis) and the bottom fraction proteins (X-axis). Proteins plotted above the dashed line represent exosome-enriched proteins, while those below the dashed line represent contaminant-enriched proteins. Importantly, many proteins that lack a transmembrane domain and are 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). Figure 3-5 As shown in the tryptic peptide coverage diagram, mass spectrometry studies led to MARCKS ( Figure 3 )、MARCKSL1( Figure 4 ) and BASP1( Figure 5 ). Since none of these proteins are predicted to have transmembrane domains, it is suggested that they are enriched as soluble proteins in the exosome lumen. In summary, these results indicate that there are many lumenal proteins that are enriched in purified exosome populations and that they can be used as payload scaffolds for the generation of engineered exosomes.

[0178] Example 2: Verification of Luminal Protein Expression

[0179] To confirm that the exosome-specific proteins identified in the mass spectrometry study were highly enriched in the exosome lumen, Western blotting was performed on total cell lysates and purified exosome populations from HEK293 cells. Fig. 6A As shown in , equal amounts of total protein from cell lysates (left) and purified exosomes (right) were loaded onto a denaturing polyacrylamide gel. MARCKS ( Figure 6B )、MARCKSL1( Figure 6C ) and BASP1( Fig.6D Western blot of 100 μg / ml luminal proteins (Figure 5A) showed that bands representing the novel luminal proteins were readily detected in exosomes but not in cell lysates, demonstrating that these proteins are highly enriched in exosomes and can be visually detected in total exosome lysates. The fact that these luminal proteins are highly expressed and enriched in exosomes provides an opportunity to generate luminal modified exosomes containing heterologous proteins fused to any of these novel proteins at high levels.

[0180] Example 3: Validation of Lumen Loading Using New Proteins as Scaffolds

[0181] To confirm the utility of MARCKS, MARCKSL1 and / or BASP1 as cavity loading scaffolds, each protein was fused to the N-terminus of GFP. In addition, 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 (well-established exosome markers) or PDGFR (a transmembrane protein with moderate exosome loading efficiency) fused to GFP were used as reference standards.

[0182] Engineered HEK293SF cells containing each expression construct were stably selected and grown to high density in 200 ml cultures. Supernatants were collected and purified by Optiprep as described in Example 1. TM Density gradient ultracentrifugation purification. The resulting GFP-containing exosomes were measured in a 96-well format. Figure 7 As shown in , the first 30 amino acids of MARCKS fused to GFP ("MARCKS (aa 1-30)") were insufficient 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 insufficient to increase exosome loading compared to CD81-GFP ("CD81"), but the full-length MARCKSL1-GFP fusion ("MARCKSL1") resulted in a signal significantly higher 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 a much greater GFP loading compared to CD81-GFP (“CD81”) or PDGFR-GFP (“pDisplay”). Fig. 9 These results suggest that BASP1 (full length or N-terminal) and full-length MARCKSL1 may be suitable scaffolds for the lumenal expression of exosomal cargo proteins.

[0183] Example 4: Identification of minimal protein sequences sufficient to carry lumenal exosome payloads

[0184] The results in Example 3 indicate that the N-terminal sequence of BASP1 is sufficient to load protein cargo into the lumen of exosomes. To determine the minimal peptide sequence with this activity, an engineered GFP loading experiment was performed by generating various BASP1 truncations fused to the N-terminus of GFP and measuring their extent of loading into exosomes. Fig.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.

[0185] BASP1 has been reported to be myristoylated, which may play a role in its localization to the exosome cavity. To test the role of myristoylation in BASP1 loading, a glycine to alanine point mutation at the predicted myristoylation site was also tested in GFP loading experiments. BASP1 1-30 (pCB 540) contained 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).

[0186] HEK293SF cells were transfected and selected in the presence of puromycin to stably express the gene encoding Fig.10 Plasmids containing each sequence in were prepared and exosomes were purified as described in Example 1. The GFP fluorescence of the purified exosomes was analyzed by nanoflow cytometry (Flow NanoAnlyzer, NanoFCM, Inc.) to determine the degree of GFP loading. Fig.11 As 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 signals, while BASP1G3A-GFP (pCB 693) showed much higher levels, indicating that the glycine at position two of BASP1 is essential for loading BASP1 fragments into exosomes, which may be due to myristoylation. BASP1 truncations pCB683-689 also showed high levels of GFP signals, while the shorter fragments pCB690-691 were similar to WT EXO. These results indicate that the nine amino acid fragment pCB689 is sufficient to drive protein cargo into the exosome lumen at very high levels.

[0187] To confirm Fig.11 The results shown in Figure 2 were analyzed by Western blotting of BASP1 fragment-GFP exosomes. Equal amounts of protein were loaded onto SDS-PAGE mini- TGX contamination-free gels (Bio-Rad, Inc.) were used to measure total exosomal protein ( Fig.12). At about 30 kDa (dashed arrow), the BASP1-GFP fragment can be detected in several lanes of the protein gel. This visualization method relies on the binding of fluorescent molecules in the gel to 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 BASP1 fragments in each lane. In order to achieve an unbiased measurement of BASP1-GFP loading into exosomes, the protein gel containing the exosome sample was stained with Coomassie blue (Invitrogen SimplyBlue SafeStain) ( Fig.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, consistent with Fig.12 The results shown in are relevant.

[0188] Western blotting with anti-FLAG antibody (M2 monoclonal antibody, Millipore-Sigma) showed that pCB540 (amino acids 1-30) and pCB683-689 had equal amounts of BASP1-GFP ( Fig.14 ), further demonstrating the ability of BASP1 to load luminal exosome cargo. The anti-FLAG signal of the shorter fragments pCB690-691 was significantly reduced or absent. BASP1G2A-GFP (pCB692) or BASP1 G2A / G3A-GFP (pCB 694) also lacked signal, while BASP1 G3A-GFP (pCB 693) was expressed at levels similar to pCB540. These results are consistent with Fig.11 The nano-flow cytometry data in the Figure 1 and Figure 2 are consistent and confirm that pCB689 is sufficient to load protein cargo into exosomes. Western blots with antibodies against the established exosomal protein Alix showed equal signal in all samples, indicating that BASP1-GFP overexpression does not disrupt the expression pattern of endogenous exosomal proteins or otherwise perturb exosome biogenesis or composition ( Fig.15 ). Together, these results indicate that a nine amino acid tag (MGGKLSKKK-SEQ ID NO: 13) can be expressed as a fusion with a heterologous protein and drive the protein into the exosome lumen. Additionally, position two of the sequence is required for exosome loading, while position three of the sequence is optional. 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.

[0189] To identify the minimal BASP1 amino acid sequence between the twelve amino acid truncations that promoted loading as shown above and the six amino acid truncations that failed to promote loading, single truncation mutants of the N-terminus of BASP1 fused to a FLAG tag and GFP were generated and stably expressed in HEK293SF cells ( Fig.16A ). Exosomes were purified from stable cell cultures as described above. BASP1 sequences of seven to twelve amino acids were able to load GFP into exosomes at high density, whereas the first six amino acids were not ( Fig. 16B These data suggest that at least one lysine residue after position six is ​​required for exosome lumen loading with the N terminus of BASP1.

[0190] The serine at position 6 of BASP1 is highly conserved among species and in MARCKS and MARCKSL1. To determine whether this amino acid is required for cargo loading into exosomes, HEK293SF cells were stably transfected with expression plasmids encoding BASP11-30-FLAG-GFP or BASP1 1-30-FLAG-GFP, including point mutants, replacing serine six with aspartic acid (S6D; polar charged substitution) or alanine (S6A; small non-polar substitution). In addition, the 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) ( Fig.17A ). BASP1 S6D completely abolished GFP loading into exosomes, whereas S6A did not alter loading. BASP1L5Q also did not affect lumen loading, indicating that the negative charge at position six disrupts loading, whereas the polar amino acid substitution at position five is well tolerated ( Fig. 17B ).

[0191] The first thirty amino acids of BASP1 contain the N-terminal leader sequence identified above, followed by a lysine-rich stretch of amino acids. To understand whether the MARCKS and MARCKSL1 N-termini can load exosomes similarly to BASP1, HEK293SF cells were stably transfected with MARCKS and MARCKSL1 full-length proteins or amino acids 1-30 fused to FLAG-GFP. The 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 with GFP, but amino acids 1-30 were not as good as the full-length proteins, suggesting that there are additional structural or sequence features required for loading in the distal regions of the MARCKS and MARCKSL1 proteins ( Fig.18). Sequence analysis of MARCKS and MARCKSL1 revealed a region 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 (PSDs) or effector domains (EDs) ( Fig.19 HEK293SF cells were stably transfected with a plasmid construct fused to amino acids 1-3 of MARCKS with the PSD domain (MG-PSD). Individual point mutations were generated at the predicted myristoylation site (MA-PSD) and position six (K6S and K6A) to determine the role of these residues in loading exosomes ( Fig. 20A ). Western blotting of purified exosomes showed that both MG-PSD and MA-PSD were unable to efficiently load exosomes compared to the positive control of BASP1 1-30. Interestingly, the K6A and K6S mutations resulted in improved loading, suggesting that the positive charge at position 6 prevents loading of exosomal cargo and that the PSD of MARCKS can functionally complement the endogenous N-terminal sequence ( Fig. 20B ). Altogether, these studies allowed the identification of several motifs that are sufficient for cargo loading into exosomes ( Fig.21 ).

[0192] The narrowest motif, Motif 1, allows a protein sequence of (M)(G)(G / A / S)(K / Q)(L / F / S / Q)(S / A)(K)(K)(SEQ ID NO:118), wherein each letter or group of letters in brackets is an amino acid position, and wherein 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). Submotifs 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)(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)(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), wherein position five cannot be a positively charged amino acid (K / R / H) and position six cannot be a negatively charged amino acid (D / E).

[0193] A broader motif Motif 2 can be expressed as (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is ​​neither (+) nor (Asp or Glu).

[0194] The most extensive motif, Motif 3, can be expressed as (M)(G)(π)(X)(Φ / π)(π)(+)(+), wherein each bracketed position represents an amino acid, and wherein π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position 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 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 native truncated sequences of BASP1. This in-depth analysis of amino acid sequence-structure-function provides new insights into the requirements for importing biologically expressed cargo into exosomes by producer cells.

[0195] Example 5: The N-terminus of BASP1 is sufficient to load different types of proteins

[0196] The results in Example 4 suggest that the N-terminus of BASP1 may be a useful engineered scaffold for generating lumen-loaded exosomes directly from production cells. To verify this hypothesis, stable HEK293SF cells were generated to express codon-optimized full-length Cas9 proteins fused to amino acids 1-30 or 1-10 of BASP1 (as described in Zetsche B, Volz SE, Zhang F. Asplit-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol. 2015 February; 33(2): 139-42). Exosomes were purified from cell culture as described above and analyzed by SDS-PAGE and Western blotting using an anti-Cas9 antibody (Abcam; Catalog No. ab191468, clone 7A9-3A3). Fig.22A As shown in , both BASP1 1-30 and 1-10 are sufficient to load Cas9 in exosomes. Recombinant Cas9 protein was used as a positive control for western blot. Densitometry quantification and comparison of various amounts of recombinant Cas9 and BASP1-Cas9 exosome lanes from western blot experiments showed that exosomes were loaded with 4-5 Cas9 molecules per exosome ( Fig. 22B). Compared to the GFP experiments shown above, the mass of this Cas9 enzyme is approximately 160 kDa, representing a significant increase in cargo size.

[0197] 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"). 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") using a second selection marker. Exosomes were purified from both transfected cell cultures and analyzed by SDS-PAGE ( Fig.23A ) and anti-ovalbumin Western blot (Abcam; catalog number ab17293, clone 6C8) were analyzed ( Fig. 23B As a control, recombinant ovalbumin (InvivoGen; catalog number vac-pova) was titrated in a separate gel. When fused to amino acids 1-10 of BASP1 as a single construct, or in combination with other overexpression plasmids (3xCD40L-PTGFRN), ovalbumin was robustly loaded into exosomes. This result suggests that exosomes can be engineered in combination with lumenal cargo and with simultaneous surface cargo (e.g. PTGFRN) from separate transcripts.

[0198] Another class of proteins that may be useful in the context of therapeutic exosomes is antibodies and antibody fragments. A single-chain camel 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. 2011 Dec 11; 19(1): 117-21) was stably expressed in HEK293SF cells as a fusion protein with amino acids 1-10 of BASP1 and a FLAG tag ("BASP1(1-10)-nanobody") or a FLAG tag alone ("nanobody") Fig.24A Analysis of purified exosomes by SDS-PAGE and anti-FLAG western blotting showed that when the nanobody was fused to the N-terminus of BASP1, there was a large enrichment of nanobodies at equal amounts of total loaded protein ( Fig. 24B ). These results indicate that using a very short protein sequence derived from the N-terminus of BASP1, a variety of protein cargoes can be expressed by producer cells and packaged into exosomes.

[0199] Example 6: The N-terminus of BASP1 can be used to load nucleic acids in the exosome cavity

[0200] Nucleic acids, and in particular RNA (e.g., mRNA, siRNA, miRNA) are an attractive class of therapeutic cargoes to be loaded into the cavity of therapeutic exosomes. Exosome loading of RNA can protect RNA from degradation in the extracellular environment, and loaded exosomes can be directed to certain cells and / or tissues through additional levels of exosome engineering (e.g., surface expression of targeting constructs). In order to understand whether the exosome proteins (or protein fragments) identified above can be used to generate exosomes loaded with mRNA, combinatorial engineered exosomes were generated. Fig.25 As shown in , amino acids 1-30 of BASP1 are expressed as fusions with FLAG and variants 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. Mutational analysis previously identified two positions in MCP that increase affinity for MS2; substitution of valine at position 29 for isoleucine (V29I; Lim & Peabody, RNA. Nucleic Acids Res. 1994 September 11; 22 (18): 3748-52) and substitution of asparagine at position 55 for lysine (N55K; Lim et al., J Biol Chem. 1994 March 25; 269 (12): 9006-10). BASP1 1-30 was fused to monomeric or dimeric MCP variants, where each MCP was V29I or doubly mutated V29I / N55K. Luciferase reporter constructs were expressed as fusions to three MS2 stem-loops from separate plasmids. Five stable HEK293SF cell lines ( luciferase-MS2 alone (#811) or in combination with each BASP1-MCP variant (#815, 817, 819, or 821) were generated. Fig.25 As an additional control, HEK293SF cells were stably transfected with FLAG-tagged BASP1 1-27. Exosomes were isolated and used The mRNA was purified by SDS-PAGE ( Fig.26A ) and anti-FLAG Western blot ( Fig.26B) analysis of purified exosomes showed equivalent amounts of total protein and comparable levels of BASP1-FLAG fusion in each exosome preparation. Importantly, BASP1-MCP fusion expressed levels comparable to BASP1 1-27FLAG fusion lacking MCP protein, indicating that addition of MCP monomer or dimer does not disrupt BASP1-mediated protein loading in exosomes.

[0201] 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)). Untransfected cells expressed lower luciferase levels than all 811-expressing cells, which expressed comparable luciferase levels ( Fig.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 binding between MCP and MS2 in promoting the loading of mRNA into exosomes ( Fig.27A , bottom). Quantification of relative mRNA between groups showed that for all BASP1-MCP fusions, enrichment was approximately 30 to 60 fold compared to 811 alone ( Fig.27B ). BASP1-MCP construct 821 containing the dimeric 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 BASP1 fragments are robust and versatile scaffold proteins for loading a variety of cargoes, including nucleic acids, into foreign body cavities.

[0202] Example 7: BASP1, MARCKS and MARCKSL1 can be used to generate surface-decorated exosomes

[0203] Results from previous experiments indicated that the full length and N-terminal regions of MARCKS, MARCKSL1, and BASP1 could 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 made for the human and mouse sequences of CD40L because the ligand does not cross-react with the homologous receptors on other species ( Fig.28 ). Exosomes were purified from HEK293SF cells stably transfected with a CD40L expression construct and incubated in mouse or human B cells. The amount of input CD40L on 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 abl19517, 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, mice ( Fig.29A ) or person ( Fig.29B ) Dose titration curves for exosomal CD40L show comparable activity between constructs on a particle-to-particle basis (left graph and table below) or compared to each other and on a CD40L molar equivalent basis for recombinant protein (right graph and table below). Comparable activity was 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, e.g., International Patent Application No. PCT / US2018 / 048026) ( Fig.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.

[0204] Example 8: Different cell types express BASP1, MARCKS and / or MARCKSL1

[0205] Cell lines from different tissue sources (HEK293, kidney; HT1080, connective tissue; K562, bone marrow; MDA-MB-231, breast; Raji, lymphoblastoid) were grown to logarithmic phase and transferred to 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 from each cell line culture were isolated and purified using the Optiprep TM Exosomes were purified by density gradient ultracentrifugation. Each purified exosome preparation was analyzed by LC-MS / MS as described above, and the number of peptide spectrum matches (PSMs) for BASP1, MARCKS and MARCKSL1, as well as two widely studied exosomal proteins (CD81 and CD9) was quantified. In most purified exosome populations, the tetraspanins CD81 and CD9 were detectable, but in some cases they were equal to or lower than luminal exosomal proteins (e.g., comparing CD9 to BASP1 or MARCKSL1) ( Fig.30 ). This finding indicates that the newly identified luminal exosome markers may be suitable fusion proteins for generating engineered exosomes from several unrelated cell lines derived from different tissues.

[0206] Example 9: Non-human cells overexpressing BASP1 produce lumen-engineered exosomes

[0207] The results in Example 8 indicate that cells of many human origins naturally express BASP1 and other novel exosomal proteins identified in Example 1. To determine whether BASP1 can be used as a universal exosomal 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"). Exosomes were purified from wild-type CHO cells and CHO cells transfected with one of the three BASP1 plasmids using the methods described in Example 1. Fig.31 As shown in AB, BASP1 and BASP1 fragment fusion proteins were successfully overexpressed in CHO cells and loaded into exosomes, as shown by stain-free PAGE ( Fig.31 A) and Western blot with antibody against FLAG ( Fig.31B). This result shows that non-human cells (such as CHO cells) can produce exosomes that overexpress human BASP1 fragments and that this 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 substances.

[0208] Example 10: Generation of Luminally Engineered Exosomes

[0209] Producer cells producing lumen-engineered exosomes are prepared by introducing exogenous sequences encoding exosomal proteins or modifications or fragments of exosomal proteins. The exosomal proteins are fusion proteins comprising the BASP1 fragments disclosed in Example 4 above and cargo proteins. Plasmids encoding exosomal proteins are transiently transfected to induce high-level expression of exosomal proteins in the exosome lumen.

[0210] The polynucleotides encoding exosome proteins, exosome protein modifications or fragments, or exogenous sequences encoding therapeutic peptides, cargo peptides or targeting moieties are stably transformed into production cells to produce cavity-engineered exosomes. The exogenous sequences encoding therapeutic peptides, cargo peptides or targeting moieties are inserted into the genomic sites encoding exosome proteins to produce fusion proteins comprising therapeutic peptides or cargo peptides attached to exosome proteins. The polynucleotides encoding modified exosome proteins are knocked into the genomic sites encoding exosome proteins.

[0211] By stably transfecting at least two polynucleotides, a production cell line is produced, each polynucleotide encoding an exosome protein, a modifier or fragment of an exosome protein, or an exogenous peptide (e.g., a targeting moiety, a therapeutic peptide). Two or more exogenous sequences are inserted into a plurality of genomic sites within or near the genomic sequence encoding the exosome protein to produce a cavity engineered exosome comprising a plurality of modified exosome proteins. Each of the various modified exosome proteins targets an exosome cavity.

[0212] Incorporated by Reference

[0213] 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 was individually indicated to be incorporated by reference for all purposes.

[0214] Equivalent solutions

[0215] 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 and methods for producing enriched exosomes. 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. Sequence Listing <110> Kodiak Biosciences <120> Compositions of engineered exosomes and methods of loading lumen exosome payloads <130> 32724-41407 / WO <140> <141> <150> 62 / 634,750 <151> 2018-02-23 <150> 62 / 587,767 <151> 2017-11-17 <160> 150 <170> PatentIn version 3.5 <210> 1 <211> 332 <212> PRT <213> Homo sapiens <400> 1 Met Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala 1 5 10 15 Glu Arg Pro Gly Glu Ala Ala Val Ala Ser Ser Pro Ser Lys Ala Asn 20 25 30 Gly Gln Glu Asn Gly His Val Lys Val Asn Gly Asp Ala Ser Pro Ala 35 40 45 Ala Ala Glu Ser Gly Ala Lys Glu Glu Leu Gln Ala Asn Gly Ser Ala 50 55 60 Pro Ala Ala Asp Lys Glu Glu Pro Ala Ala Ala Gly Ser Gly Ala Ala 65 70 75 80 Ser Pro Ser Ala Ala Glu Lys Gly Glu Pro Ala Ala Ala Ala Ala Pro 85 90 95 Glu Ala Gly Ala Ser Pro Val Glu Lys Glu Ala Pro Ala Glu Gly Glu 100 105 110 Ala Ala Glu Pro Gly Ser Pro Thr Ala Ala Glu Gly Glu Ala Ala Ser 115 120 125 Ala Ala Ser Ser Thr Ser Ser Pro Lys Ala Glu Asp Gly Ala Thr Pro 130 135 140 Ser Pro Ser Asn Glu Thr Pro Lys Lys Lys Lys Lys Arg Phe Ser Phe 145 150 155 160 Lys Lys Ser Phe Lys Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys 165 170 175 Glu Ala Gly Glu Gly Gly Glu Ala Glu Ala Pro Ala Ala Glu Gly Gly 180 185 190 Lys Asp Glu Ala Ala Gly Gly Ala Ala Ala Ala Ala Ala Glu Ala Gly 195 200 205 Ala Ala Ser Gly Glu Gln Ala Ala Ala Pro Gly Glu Glu Ala Ala Ala 210 215 220 Gly Glu Glu Gly Ala Ala Gly Gly Asp Pro Gln Glu Ala Lys Pro Gln 225 230 235 240 Glu Ala Ala Val Ala Pro Glu Lys Pro Pro Ala Ser Asp Glu Thr Lys 245 250 255 Ala Ala Glu Glu Pro Ser Lys Val Glu Glu Lys Lys Ala Glu Glu Ala 260 265 270 Gly Ala Ser Ala Ala Ala Cys Glu Ala Pro Ser Ala Ala Gly Pro Gly 275 280 285 Ala Pro Pro Glu Gln Glu Ala Ala Pro Ala Glu Glu Pro Ala Ala Ala 290 295 300 Ala Ala Ser Ser Ala Cys Ala Ala Pro Ser Gln Glu Ala Gln Pro Glu 305 310 315 320 Cys Ser Pro Glu Ala Pro Pro Ala Glu Ala Ala Glu 325 330 <210> 2 <211> 195 <212> PRT <213> Homo sapiens <400> 2 Met Gly Ser Gln Ser Ser Lys Ala Pro Arg Gly Asp Val Thr Ala Glu 1 5 10 15 Glu Ala Ala Gly Ala Ser Pro Ala Lys Ala Asn Gly Gln Glu Asn Gly 20 25 30 His Val Lys Ser Asn Gly Asp Leu Ser Pro Lys Gly Glu Gly Glu Ser 35 40 45 Pro Pro Val Asn Gly Thr Asp Glu Ala Ala Gly Ala Thr Gly Asp Ala 50 55 60 Ile Glu Pro Ala Pro Pro Ser Gln Gly Ala Glu Ala Lys Gly Glu Val 65 70 75 80 Pro Pro Lys Glu Thr Pro Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys 85 90 95 Pro Phe Lys Leu Ser Gly Leu Ser Phe Lys Arg Asn Arg Lys Glu Gly 100 105 110 Gly Gly Asp Ser Ser Ala Ser Ser Pro Thr Glu Glu Glu Gln Glu Gln 115 120 125 Gly Glu Ile Gly Ala Cys Ser Asp Glu Gly Thr Ala Gln Glu Gly Lys 130 135 140 Ala Ala Ala Thr Pro Glu Ser Gln Glu Pro Gln Ala Lys Gly Ala Glu 145 150 155 160 Ala Ser Ala Ala Ser Glu Glu Glu Ala Gly Pro Gln Ala Thr Glu Pro 165 170 175 Ser Thr Pro Ser Gly Pro Glu Ser Gly Pro Thr Pro Ala Ser Ala Glu 180 185 190 Gln Asn Glu 195 <210> 3 <211> 227 <212> PRT <213> Homo sapiens <400> 3 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Thr Glu 20 25 30 Glu Glu Gly Thr Pro Lys Glu Ser Glu Pro Gln Ala Ala Ala Glu Pro 35 40 45 Ala Glu Ala Lys Glu Gly Lys Glu Lys Pro Asp Gln Asp Ala Glu Gly 50 55 60 Lys Ala Glu Glu Lys Glu Gly Glu Lys Asp Ala Ala Ala Ala Lys Glu 65 70 75 80 Glu Ala Pro Lys Ala Glu Pro Glu Lys Thr Glu Gly Ala Ala Glu Ala 85 90 95 Lys Ala Glu Pro Pro Lys Ala Pro Glu Gln Glu Gln Ala Ala Pro Gly 100 105 110 Pro Ala Ala Gly Gly Glu Ala Pro Lys Ala Ala Glu Ala Ala Ala Ala 115 120 125 Pro Ala Glu Ser Ala Ala Pro Ala Ala Gly Glu Glu Pro Ser Lys Glu 130 135 140 Glu Gly Glu Pro Lys Lys Thr Glu Ala Pro Ala Ala Pro Ala Ala Gln 145 150 155 160 Glu Thr Lys Ser Asp Gly Ala Pro Ala Ser Asp Ser Lys Pro Gly Ser 165 170 175 Ser Glu Ala Ala Pro Ser Ser Lys Glu Thr Pro Ala Ala Thr Glu Ala 180 185 190 Pro Ser Ser Thr Pro Lys Ala Gln Gly Pro Ala Ala Ser Ala Glu Glu 195 200 205 Pro Lys Pro Val Glu Ala Pro Ala Ala Asn Ser Asp Gln Thr Val Thr 210 215 220 Val Lys Glu 225 <210> 4 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 4 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala 20 25 30 <210> 5 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 5 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu 20 25 <210> 6 <211> twenty four <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 6 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys 20 <210> 7 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 7 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu 20 <210> 8 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 8 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys <210> 9 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 9 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn 1 5 10 15 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 10 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr 1 5 10 <210> 11 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 11 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Lys Gly 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 12 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys 1 5 10 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 13 Met Gly Gly Lys Leu Ser Lys Lys Lys 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 14 Met Gly Gly Lys Leu Ser Lys Lys 1 5 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 15 Met Gly Gly Lys Leu Ser Lys 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 16 Met Gly Gly Lys Leu Ala Lys Lys 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 17 Met Gly Gly Lys Phe Ser Lys Lys 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 18 Met Gly Gly Lys Phe Ala Lys Lys 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 19 Met Gly Gly Lys Ser Ser Lys Lys 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 20 Met Gly Gly Lys Ser Ala Lys Lys 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty one Met Gly Gly Lys Gln Ser Lys Lys 1 5 <210> twenty two <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty two Met Gly Gly Lys Gln Ala Lys Lys 1 5 <210> twenty three <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty three Met Gly Gly Gln Leu Ser Lys Lys 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty four Met Gly Gly Gln Leu Ala Lys Lys 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 25 Met Gly Gly Gln Phe Ser Lys Lys 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 26 Met Gly Gly Gln Phe Ala Lys Lys 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 27 Met Gly Gly Gln Ser Ser Lys Lys 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 28 Met Gly Gly Gln Ser Ala Lys Lys 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 29 Met Gly Gly Gln Gln Ser Lys Lys 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 30 Met Gly Gly Gln Gln Ala Lys Lys 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 31 Met Gly Ala Lys Leu Ser Lys Lys 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 32 Met Gly Ala Lys Leu Ala Lys Lys 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 33 Met Gly Ala Lys Phe Ser Lys Lys 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 34 Met Gly Ala Lys Phe Ala Lys Lys 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 35 Met Gly Ala Lys Ser Ser Lys Lys 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 36 Met Gly Ala Lys Ser Ala Lys Lys 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 37 Met Gly Ala Lys Gln Ser Lys Lys 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 38 Met Gly Ala Lys Gln Ala Lys Lys 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 39 Met Gly Ala Gln Leu Ser Lys Lys 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 40 Met Gly Ala Gln Leu Ala Lys Lys 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 41 Met Gly Ala Gln Phe Ser Lys Lys 1 5 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 42 Met Gly Ala Gln Phe Ala Lys Lys 1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 43 Met Gly Ala Gln Ser Ser Lys Lys 1 5 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 44 Met Gly Ala Gln Ser Ala Lys Lys 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 45 Met Gly Ala Gln Gln Ser Lys Lys 1 5 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 46 Met Gly Ala Gln Gln Ala Lys Lys 1 5 <210> 47 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 47 Met Gly Ser Lys Leu Ser Lys Lys 1 5 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 48 Met Gly Ser Lys Leu Ala Lys Lys 1 5 <210> 49 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 49 Met Gly Ser Lys Phe Ser Lys Lys 1 5 <210> 50 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 50 Met Gly Ser Lys Phe Ala Lys Lys 1 5 <210> 51 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 51 Met Gly Ser Lys Ser Ser Lys Lys 1 5 <210> 52 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 52 Met Gly Ser Lys Ser Ala Lys Lys 1 5 <210> 53 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 53 Met Gly Ser Lys Gln Ser Lys Lys 1 5 <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 54 Met Gly Ser Lys Gln Ala Lys Lys 1 5 <210> 55 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 55 Met Gly Ser Gln Leu Ser Lys Lys 1 5 <210> 56 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 56 Met Gly Ser Gln Leu Ala Lys Lys 1 5 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 57 Met Gly Ser Gln Phe Ser Lys Lys 1 5 <210> 58 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 58 Met Gly Ser Gln Phe Ala Lys Lys 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 59 Met Gly Ser Gln Ser Ser Lys Lys 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 60 Met Gly Ser Gln Ser Ala Lys Lys 1 5 <210> 61 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 61 Met Gly Ser Gln Gln Ser Lys Lys 1 5 <210> 62 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 62 Met Gly Ser Gln Gln Ala Lys Lys 1 5 <210> 63 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 63 Met Gly Gly Lys Leu Ala Lys 1 5 <210> 64 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 64 Met Gly Gly Lys Phe Ser Lys 1 5 <210> 65 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 65 Met Gly Gly Lys Phe Ala Lys 1 5 <210> 66 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 66 Met Gly Gly Lys Ser Ser Lys 1 5 <210> 67 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 67 Met Gly Gly Lys Ser Ala Lys 1 5 <210> 68 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 68 Met Gly Gly Lys Gln Ser Lys 1 5 <210> 69 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 69 Met Gly Gly Lys Gln Ala Lys 1 5 <210> 70 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 70 Met Gly Gly Gln Leu Ser Lys 1 5 <210> 71 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 71 Met Gly Gly Gln Leu Ala Lys 1 5 <210> 72 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 72 Met Gly Gly Gln Phe Ser Lys 1 5 <210> 73 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 73 Met Gly Gly Gln Phe Ala Lys 1 5 <210> 74 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 74 Met Gly Gly Gln Ser Ser Lys 1 5 <210> 75 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 75 Met Gly Gly Gln Ser Ala Lys 1 5 <210> 76 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 76 Met Gly Gly Gln Gln Ser Lys 1 5 <210> 77 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 77 Met Gly Gly Gln Gln Ala Lys 1 5 <210> 78 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 78 Met Gly Ala Lys Leu Ser Lys 1 5 <210> 79 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 79 Met Gly Ala Lys Leu Ala Lys 1 5 <210> 80 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 80 Met Gly Ala Lys Phe Ser Lys 1 5 <210> 81 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 81 Met Gly Ala Lys Phe Ala Lys 1 5 <210> 82 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 82 Met Gly Ala Lys Ser Ser Lys 1 5 <210> 83 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 83 Met Gly Ala Lys Ser Ala Lys 1 5 <210> 84 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 84 Met Gly Ala Lys Gln Ser Lys 1 5 <210> 85 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 85 Met Gly Ala Lys Gln Ala Lys 1 5 <210> 86 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 86 Met Gly Ala Gln Leu Ser Lys 1 5 <210> 87 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 87 Met Gly Ala Gln Leu Ala Lys 1 5 <210> 88 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 88 Met Gly Ala Gln Phe Ser Lys 1 5 <210> 89 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 89 Met Gly Ala Gln Phe Ala Lys 1 5 <210> 90 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 90 Met Gly Ala Gln Ser Ser Lys 1 5 <210> 91 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 91 Met Gly Ala Gln Ser Ala Lys 1 5 <210> 92 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 92 Met Gly Ala Gln Gln Ser Lys 1 5 <210> 93 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 93 Met Gly Ala Gln Gln Ala Lys 1 5 <210> 94 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 94 Met Gly Ser Lys Leu Ser Lys 1 5 <210> 95 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 95 Met Gly Ser Lys Leu Ala Lys 1 5 <210> 96 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 96 Met Gly Ser Lys Phe Ser Lys 1 5 <210> 97 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 97 Met Gly Ser Lys Phe Ala Lys 1 5 <210> 98 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 98 Met Gly Ser Lys Ser Ser Lys 1 5 <210> 99 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 99 Met Gly Ser Lys Ser Ala Lys 1 5 <210> 100 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 100 Met Gly Ser Lys Gln Ser Lys 1 5 <210> 101 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 101 Met Gly Ser Lys Gln Ala Lys 1 5 <210> 102 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 102 Met Gly Ser Gln Leu Ser Lys 1 5 <210> 103 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 103 Met Gly Ser Gln Leu Ala Lys 1 5 <210> 104 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 104 Met Gly Ser Gln Phe Ser Lys 1 5 <210> 105 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 105 Met Gly Ser Gln Phe Ala Lys 1 5 <210> 106 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 106 Met Gly Ser Gln Ser Ser Lys 1 5 <210> 107 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 107 Met Gly Ser Gln Ser Ala Lys 1 5 <210> 108 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 108 Met Gly Ser Gln Gln Ser Lys 1 5 <210> 109 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 109 Met Gly Ser Gln Gln Ala Lys 1 5 <210> 110 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 110 Met Gly Ala Lys Leu Ser Lys Lys Lys 1 5 <210> 111 <211> 167 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 111 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Ser Gly 20 25 30 Gly Ser Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly 35 40 45 Ser Gly Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly 50 55 60 Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile 65 70 75 80 Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr 85 90 95 Cys Ser Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys 100 105 110 Val Glu Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu 115 120 125 Thr Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys 130 135 140 Ala Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile 145 150 155 160 Ala Ala Asn Ser Gly Ile Tyr 165 <210> 112 <211> 167 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 112 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Ser Gly 20 25 30 Gly Ser Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly 35 40 45 Ser Gly Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly 50 55 60 Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile 65 70 75 80 Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr 85 90 95 Cys Ser Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys 100 105 110 Val Glu Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu 115 120 125 Thr Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys 130 135 140 Ala Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile 145 150 155 160 Ala Ala Asn Ser Gly Ile Tyr 165 <210> 113 <211> 296 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic polypeptide <400> 113 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Ser Gly 20 25 30 Gly Ser Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly 35 40 45 Ser Gly Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly 50 55 60 Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile 65 70 75 80 Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr 85 90 95 Cys Ser Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys 100 105 110 Val Glu Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu 115 120 125 Thr Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys 130 135 140 Ala Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile 145 150 155 160 Ala Ala Asn Ser Gly Ile Tyr Gly Ser Gly Gly Ser Gly Gly Ser Gly 165 170 175 Gly Ser Gly Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn 180 185 190 Gly Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly 195 200 205 Ile Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val 210 215 220 Thr Cys Ser Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile 225 230 235 240 Lys Val Glu Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu 245 250 255 Leu Thr Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val 260 265 270 Lys Ala Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala 275 280 285 Ile Ala Ala Asn Ser Gly Ile Tyr 290 295 <210> 114 <211> 296 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 114 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Ser Gly 20 25 30 Gly Ser Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly 35 40 45 Ser Gly Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly 50 55 60 Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile 65 70 75 80 Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr 85 90 95 Cys Ser Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys 100 105 110 Val Glu Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu 115 120 125 Thr Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys 130 135 140 Ala Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile 145 150 155 160 Ala Ala Asn Ser Gly Ile Tyr Gly Ser Gly Gly Ser Gly Gly Ser Gly 165 170 175 Gly Ser Gly Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn 180 185 190 Gly Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly 195 200 205 Ile Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val 210 215 220 Thr Cys Ser Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile 225 230 235 240 Lys Val Glu Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu 245 250 255 Leu Thr Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val 260 265 270 Lys Ala Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala 275 280 285 Ile Ala Ala Asn Ser Gly Ile Tyr 290 295 <210> 115 <211> 680 <212> RNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 115 augaagccca ccgagaacaa cgaagacuuc aacaucgugg ccguggccag caacuucgcg 60 accacggauc ucgaugcuga ccgcgggaag uugcccggca agaagcugcc gcuggaggug 120 cucaaagagu uggaagccaa ugcccggaaa gcuggcugca ccaggggcug ucugaucugc 180 cugucccaca ucaagugcac gcccaagaug aagaaguuca ucccaggacg cugccacacc 240 uacgaaggcg acaaagaguc cgcacagggc ggcauaggcg aggcgaucgu cgacauuccu 300 gagauuccug gguucaagga cuuggagccc uuggagcagu ucaucgcaca ggucgaucug 360 uguguggacu gcacaacugg cugccucaaa gggcuugcca acgugcagug uucugaccug 420 cucaagaagu ggcugccgca acgcugugcg accuuugcca gcaagaucca gggccaggug 480 gacaagauca agggggccgg uggugacuaa ggauccaucg auaagcuuca ucgaaacaug 540 aggaucaccc auaucugcag ucgacaucga aacaugagga ucaccaugu cugcagucga 600 caucgaaaca ugaggaucac ccaugucugc agucgacauc gaaacaugag gaucaccccau 660 gucugcaguc gacaucgaaa 680 <210> 116 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (3) <223> Any amino acid <400> 116 Met Gly Xaa Lys Leu Ser Lys Lys Lys 1 5 <210> 117 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (3) <223> Any amino acid <220> <223> For a detailed description of substitutions and preferred embodiments, see the filed specification. <400> 117 Met Gly Xaa Lys Leu Ser Lys Lys Lys 1 5 <210> 118 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (3) <223> Gly, Ala or Ser <220> <221> MOD_RES <222> (4) <223> Lys or Gln <220> <221> MOD_RES <222> (5) <223> Leu, Phe, Ser or Gln <220> <221> MOD_RES <222> (6) <223> Ser or Ala <220> <223> For a detailed description of substitutions and preferred embodiments, see the filed specification. <400> 118 Met Gly Xaa Xaa Xaa Xaa Lys Lys 1 5 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic primers <400> 119 tggaggtgct caaagagttg 20 <210> 120 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic primers <400> 120 ttgggcgtgc acttgat 17 <210> 121 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic probes <400> 121 gggcattggc ttc 13 <210> 122 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 122 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Ser Ala 20 25 30 Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly 35 40 45 Gly Ser Val Ser Lys Gly Glu Glu Leu Phe Thr Gly 50 55 60 <210> 123 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 123 Met Ala Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Ser Ala 20 25 30 Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly 35 40 45 Gly Ser Val Ser Lys Gly Glu Glu Leu Phe Thr Gly 50 55 60 <210> 124 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 124 Met Gly Ala Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Ser Ala 20 25 30 Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly 35 40 45 Gly Ser Val Ser Lys Gly Glu Glu Leu Phe Thr Gly 50 55 60 <210> 125 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 125 Met Ala Ala Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Ser Ala 20 25 30 Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly 35 40 45 Gly Ser Val Ser Lys Gly Glu Glu Leu Phe Thr Gly 50 55 60 <210> 126 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 126 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Ser Ala Gly Gly Gly 20 25 30 Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val 35 40 45 Ser Lys Gly Glu Glu Leu Phe Thr Gly 50 55 <210> 127 <211> 54 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 127 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Ser Ala Gly Gly Gly Gly Ser Asp 20 25 30 Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val Ser Lys Gly 35 40 45 Glu Glu Leu Phe Thr Gly 50 <210> 128 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 128 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Ser Ala Gly Gly Gly Gly Ser Asp Tyr Lys Asp 20 25 30 Asp Asp Asp Lys Gly Gly Gly Gly Ser Val Ser Lys Gly Glu Glu Leu 35 40 45 Phe Thr Gly 50 <210> 129 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 129 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ser Ala Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp 20 25 30 Lys Gly Gly Gly Gly Ser Val Ser Lys Gly Glu Glu Leu Phe Thr Gly 35 40 45 <210> 130 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 130 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Ser 1 5 10 15 Ala Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly 20 25 30 Gly Gly Ser Val Ser Lys Gly Glu Glu Leu Phe Thr Gly 35 40 45 <210> 131 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 131 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Ser Ala Gly Gly 1 5 10 15 Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser 20 25 30 Val Ser Lys Gly Glu Glu Leu Phe Thr Gly 35 40 <210> 132 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 132 Met Gly Gly Lys Leu Ser Lys Lys Lys Ser Ala Gly Gly Gly Gly Ser 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val Ser Lys 20 25 30 Gly Glu Glu Leu Phe Thr Gly 35 <210> 133 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 133 Met Gly Gly Lys Leu Ser Ser Ala Gly Gly Gly Gly Ser Asp Tyr Lys 1 5 10 15 Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val Ser Lys Gly Glu Glu 20 25 30 Leu Phe Thr Gly 35 <210> 134 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 134 Met Gly Gly Ser Ala Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp 1 5 10 15 Asp Lys Gly Gly Gly Gly Ser Val Ser Lys Gly Glu Glu Leu Phe Thr 20 25 30 Gly <210> 135 <211> 54 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 135 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Ser Ala 20 25 30 Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly 35 40 45 Gly Ser Val Ser Lys Gly 50 <210> 136 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 136 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Ser Ala Gly Gly 1 5 10 15 Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser 20 25 30 Val Ser Lys Gly 35 <210> 137 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 137 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Ser Ala Gly Gly Gly 1 5 10 15 Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val 20 25 30 Ser Lys Gly 35 <210> 138 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 138 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Ser Ala Gly Gly Gly Gly 1 5 10 15 Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val Ser 20 25 30 Lys Gly <210> 139 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 139 Met Gly Gly Lys Leu Ser Lys Lys Lys Ser Ala Gly Gly Gly Gly Ser 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val Ser Lys 20 25 30 Gly <210> 140 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 140 Met Gly Gly Lys Leu Ser Lys Lys Ser Ala Gly Gly Gly Gly Ser Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val Ser Lys Gly 20 25 30 <210> 141 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 141 Met Gly Gly Lys Leu Ser Lys Ser Ala Gly Gly Gly Gly Ser Asp Tyr 1 5 10 15 Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val Ser Lys Gly 20 25 30 <210> 142 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 142 Met Gly Gly Lys Leu Ser Ser Ala Gly Gly Gly Gly Ser Asp Tyr Lys 1 5 10 15 Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Val Ser Lys Gly 20 25 30 <210> 143 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 143 Met Gly Gly Lys Leu Asp Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala 20 25 30 <210> 144 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 144 Met Gly Gly Lys Leu Ala Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala 20 25 30 <210> 145 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 145 Met Gly Gly Lys Gln Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp 1 5 10 15 Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala 20 25 30 <210> 146 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 146 Met Gly Ala Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe 1 5 10 15 Lys Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys Glu Ala 20 25 30 <210> 147 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 147 Met Ala Ala Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe 1 5 10 15 Lys Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys Glu Ala 20 25 30 <210> 148 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 148 Met Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe 1 5 10 15 Lys Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys Glu Ala 20 25 30 <210> 149 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 149 Met Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Pro Phe 1 5 10 15 Lys Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys Glu Ala 20 25 30 <210> 150 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 150 Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Ser Ala Gly Gly Ser Gly 1 5 10 15 Gly Ser Thr Ser Gly Ser Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly 20 25 30 Ser Gly Phe Glu Met Asp Gln Val Gln Leu Val Glu Ser Gly Gly Ala 35 40 45 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 50 55 60 Phe Pro Val Asn Arg Tyr Ser Met Arg Trp Tyr Arg Gln Ala Pro Gly 65 70 75 80 Lys Glu Arg Glu Trp Val Ala Gly Met Ser Ser Ala Gly Asp Arg Ser 85 90 95 Ser Tyr Glu Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp 100 105 110 Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 115 120 125 Thr Ala Val Tyr Tyr Cys Asn Val Asn Val Gly Phe Glu Tyr Trp Gly 130 135 140 Gln Gly Thr Gln Val Thr Val Ser Ser 145 150

Claims

1. An exosome comprising a fusion protein, wherein the fusion protein comprises a biologically active payload and a scaffold protein, wherein the scaffold protein consists of: (i) MARCKSL1 consisting of a sequence according to SEQ ID NO: 2, or (ii) BASP1 consisting of a sequence according to any one of SEQ ID NOs: 3-15, wherein the biologically active payload is selected from the group consisting of nucleotides and proteins.

2. The exosome of claim 1, wherein the fusion protein is present in the exosome at a higher density than a different fusion protein in a different exosome, wherein the different fusion protein comprises a conventional exosomal protein or a variant thereof.

3. The exosome of claim 2, wherein the conventional exosomal protein is selected from the group consisting of CD9, CD63, CD81, PDGFR, GPI anchor protein, lactadherin, LAMP2, LAMP2B and fragments thereof.

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

5. The exosome of claim 4, wherein the cell is a HEK293 cell.

6. The exosome of claim 4, wherein the cell comprises a plasmid comprising the exogenous sequence.

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

8. The exosome of claim 7, wherein the exogenous sequence is inserted into a genomic site located 3' or 5' relative to the genomic sequence encoding MARCKSL1 or BASP1.

9. The exosome of claim 7, wherein the exogenous sequence is inserted into a genomic sequence encoding MARCKSL1 or BASP1.

10. The exosome of claim 1, wherein the biologically active payload comprises a fusion protein of a therapeutic peptide.

11. The exosome of claim 1, wherein the biologically active payload comprises a therapeutic peptide selected from the group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to a therapeutic compound.

12. The exosome of claim 1, wherein the biologically active payload is an antibody or a fragment or modification thereof.

13. The exosome of claim 1, wherein the biologically active payload is an enzyme, a ligand, a receptor, a transcription factor, or a fragment or modification thereof.

14. The exosome of claim 1, wherein the biologically active payload is an antimicrobial peptide or a fragment or modification thereof.

15. A cell population for producing the exosomes according to any one of claims 1 to 14. The cell population of claim 15 , comprising an exogenous sequence encoding the scaffold protein of claim 1 .

17. The cell population of claim 15 or 16, wherein the exogenous sequence is inserted into a genomic sequence encoding MARCKSL1, or BASP1, wherein the exogenous sequence and the genomic sequence encode the scaffold protein.

18. The cell population of claim 15 or 16, wherein the exogenous sequence is in a plasmid.

19. The cell population of claim 16, wherein the exogenous sequence encodes a biologically active payload comprising a therapeutic peptide.

20. The cell population of claim 19, wherein the biologically active payload is selected from the group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to a therapeutic compound.

21. The cell population of claim 19, wherein the biologically active payload is an antibody or a fragment or modification thereof.

22. The cell population of claim 19, wherein the biologically active payload is an enzyme, a ligand, a receptor, a transcription factor, or a fragment or modification thereof.

23. The cell population of claim 19, wherein the therapeutic peptide is an antimicrobial peptide or a fragment or modification thereof.

24. The cell population of claim 16, wherein the exogenous sequence encodes a targeting moiety.

25. The cell population of claim 24, wherein the targeting moiety is specific for an organ, tissue or cell.

Images