PREPARATION OF THERAPEUTIC EXOSOMES USING MEMBRANE PROTEINS
The present invention relates to methods of preparing a therapeutic exosome using a protein newly-identified to be enriched on the surface of exosomes. Specifically, the present invention provides methods of using the proteins for affinity purification of exosomes. It also provides methods of localizing a therapeutic peptide on exosomes, and targeting exosomes to a specific organ, tissue or cell by using the proteins. The methods involve generation of surface-engineered exosomes that include one or more of the exosome proteins at higher density, or a variant or a fragment of the exosome protein.
1 . A method of isolating an exosome, comprising the steps of:
providing a sample comprising the exosome;
contacting the sample with a binding agent having affinity to a target protein, wherein the target protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment or a variant thereof; and
isolating the exosome based on a binding between the target protein and the binding agent.
2 . The method of claim 1 , wherein said sample is obtained from a cell grown in vitro, optionally wherein the cell is an HEK293 cell.
3 . The method of claim 1 , wherein said sample is obtained from a body fluid of a subject.
4 . The method of claim 2 , wherein the cell is genetically modified to express the target protein.
5 . The method of claim 2 or 4 , wherein the cell comprises an expression plasmid encoding the target protein.
6 . The method of claim 4 , wherein the cell is genetically modified to comprise an exogenous sequence expressing a tag having affinity to the binding agent, wherein the exogenous sequence is inserted into a genome of the cell.
7 . The method of claim 6 , wherein the exogenous sequence is inserted in a genomic site located at 3′ or 5′ end of an endogenous sequence encoding PTGFRN, BSG, IGSF2, IGSF3, ITGB1, ITGA4, SLC3A2 or ATP transporter.
8 . The method of claim 6 , wherein the exogenous sequence is inserted in a genomic site located within an endogenous sequence encoding PTGFRN, BSG, IGSF2, IGSF3, ITGB1, ITGA4, SLC3A2 or ATP transporter.
9 . The method of any of claims 7-8 , wherein the target protein is a fusion protein comprising the tag, and PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment or a variant thereof.
10 . The method of any of claims 1-9 , wherein the cell is genetically modified to have a reduced expression of ADAM10.
11 . The method of any of claims 1-10 , wherein the exosome comprises the target protein.
12 . The method of any of claims 1-11 , wherein the target protein is selected from PTGFRN, BSG, IGSF3, ITGB1, ITGA4, SLC3A2 and ATP transporter.
13 . The method of any of claims 1-11 , wherein the target protein comprises a fragment or a variant of PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2 or ATP transporter.
14 . The method of claim 13 , wherein the target protein comprises a polypeptide of SEQ ID NO: 33.
15 . The method of any of claims 13-14 , wherein the target protein is a fusion protein comprising PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment or a variant thereof, and an affinity tag, wherein the affinity tag has affinity to the binding agent.
16 . The method of claim 15 , wherein the binding agent comprises an immunoglobulin, a protein, a peptide, or a small molecule.
17 . The method of any of claims 1-16 , wherein the binding agent is attached to a solid support, optionally wherein the solid support comprises a porous agarose bead, a microtiter plate, a magnetic bead, or a membrane.
18 . The method of claim 17 , wherein the solid support forms a chromatography column.
19 . The method of claim 18 , wherein the step of contacting the sample with the binding agent is performed by applying the sample to the chromatography column.
20 . The method of any of claims 1-19 , further comprising the steps of:
contacting a subset of the sample with a different binding agent having affinity to a different target protein; and
isolating the exosome based on a binding between the different target protein and the different binding agent.
21 . The method of any of claims 1-20 , wherein the different target protein comprises PTGFRN, BSG, IGSF3, IGSF2, ATP transporter or a fragment or a variant thereof.
22 . The method of any of claims 1-21 , wherein the different target protein comprises PTGFRN or a fragment or a variant thereof.
23 . The method of claim 22 , wherein the different target protein comprises a polypeptide of SEQ ID NO: 33.
24 . An exosome produced by the method of any of claims 1-23 .
25 . A pharmaceutical composition comprising the exosome of claim 24 and an excipient.
26 . The pharmaceutical composition of claim 25 , wherein the pharmaceutical composition comprises a lower concentration of macromolecules than the sample, wherein the macromolecules are nucleic acids, contaminant proteins, lipids, carbohydrates, metabolites, or a combination thereof.
27 . The pharmaceutical composition of claim 26 , wherein the pharmaceutical composition is substantially free of the macromolecules.
28 . An exosome comprising a target protein, wherein at least a part of the target protein is expressed from an exogenous sequence, and the target protein comprises PTGFRN, BSG, IGSF3, IGSF8, IGSF2, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment or a variant thereof.
29 . The exosome of claim 28 , wherein the target protein is present on the surface of the exosome at a higher density than a different target protein of a different exosome, wherein the different target protein comprises a conventional exosome protein or a variant thereof.
30 . The exosome of claim 29 , wherein the conventional exosome protein is selected from the group consisting of CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin, LAMP2, LAMP2B, and a fragment thereof.
31 . The exosome of any of claims 28-30 , wherein the target protein comprises a polypeptide of SEQ ID NO: 33.
32 . The exosome of any of claims 28-31 , isolated based on a binding between the target protein and a binding agent.
33 . The exosome of any of claims 28-32 , produced from a cell genetically modified to comprise the exogenous sequence, optionally wherein the cell is an HEK293 cell.
34 . The exosome of claim 33 , wherein the cell is genetically modified to have a reduced expression of ADAM10.
35 . The exosome of any of claims 33-34 , wherein the cell comprises a plasmid comprising the exogenous sequence.
36 . The exosome of any of claims 33-35 , wherein the cell comprises the exogenous sequence inserted into a genome of the cell.
37 . The exosome of claim 36 , wherein the exogenous sequence is inserted into a genomic site located 3′ or 5′ end of a genomic sequence encoding PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2 or ATP transporter.
38 . The exosome of claim 36 , wherein the exogenous sequence is inserted into a genomic sequence encoding PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2 or ATP transporter.
39 . The exosome of any of claims 28-38 , wherein the target protein is a fusion protein comprising PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment or a variant thereof, and an affinity tag, wherein the affinity tag has affinity to the binding agent.
40 . The exosome of any of claims 28-38 , wherein the target protein is a fusion protein comprising PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment or a variant thereof, and a therapeutic peptide.
41 . The exosome of claim 40 , wherein the therapeutic peptide is selected from the group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to a therapeutic compound.
42 . The exosome of claim 40 , wherein the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, and small molecules.
43 . The exosome of claim 40 , wherein the therapeutic peptide is an antibody or a fragment or a variant thereof.
44 . The exosome of claim 40 , wherein the therapeutic peptide is an enzyme, a ligand, a receptor, or a fragment or a variant thereof.
45 . The exosome of claim 40 , wherein the therapeutic peptide is an antimicrobial peptide or a fragment or a variant thereof.
46 . The exosome of any of claims 28-38 , wherein the target protein is a fusion protein comprising PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment or a variant thereof, and a targeting moiety.
47 . The exosome of claim 46 , wherein the targeting moiety is specific to an organ, a tissue, or a cell.
48 . The exosome of any of claims 28-47 , further comprising a different target protein, wherein the different target protein comprises PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment or a variant thereof.
49 . The exosome of claim 48 , isolated based on a binding between the different target protein and a different binding agent.
50 . A pharmaceutical composition comprising the exosome of any of claims 28-49 and an excipient.
51 . The pharmaceutical composition of claim 50 , substantially free of macromolecules, wherein the macromolecules are selected from nucleic acids, contaminant proteins, lipids, carbohydrates, metabolites, and a combination thereof.
52 . A cell line for producing the exosome of any of claims 28-49 .
53 . A cell line for producing exosomes, comprising an exogenous sequence inserted into a genomic sequence encoding PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2, or ATP transporter, wherein the exogenous sequence and the genomic sequence encodes a fusion protein.
54 . The cell line of claim 53 , wherein the exogenous sequence encodes an affinity tag.
55 . The cell line of claim 53 , wherein the exogenous sequence encodes a therapeutic peptide.
56 . The cell line of claim 55 , wherein the therapeutic peptide is selected from the group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to a therapeutic compound.
57 . The cell line of claim 55 , wherein the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, and small molecules.
58 . The cell line of claim 55 , wherein the therapeutic peptide is an antibody or a fragment or a variant thereof.
59 . The cell line of claim 55 , wherein the therapeutic peptide is an enzyme, a ligand, a receptor, or a fragment or a variant thereof.
60 . The cell line of claim 55 , wherein the therapeutic peptide is an antimicrobial peptide or a fragment or a variant thereof.
61 . The cell line of claim 53 , wherein the exogenous sequence encodes a targeting moiety.
62 . The cell line of claim 61 , wherein the targeting moiety is specific to an organ, a tissue, or a cell.
63 . The cell line of any of claims 53-62 , wherein the cell line is genetically modified to have a reduced expression of ADAM10.
64 . An exosome produced from the cell line of any of claims 53-63 .
65 . The exosome of claim 64 , wherein the exosome includes the fusion protein on the surface at a higher density than a different fusion protein on the surface of a different exosome, wherein the different exosome is produced from a different cell line comprising the exogenous sequence inserted into a different genomic sequence encoding a conventional exosome protein, wherein the exogenous sequence and the different genomic sequence encodes the different fusion protein.
66 . The exosome of claim 65 , wherein the conventional exosome protein is selected from the group consisting of CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin, LAMP2, LAMP2B, and a fragment thereof.
67 . A method of isolating a non-exosomal material, comprising the steps of:
providing a sample comprising an exosome and the non-exosome material;
contacting the sample with a binding agent having affinity to a target protein, wherein the target protein comprises PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment or a variant thereof, thereby inducing the exosome to bind to the binding agent; and
isolating the non-exosome material.
68 . The method of claim 67 , wherein the non-exosomal material is virus or a protein.
69 . The method of claim 68 , wherein the non-exosomal material is lentivirus, retrovirus, adeno-associated virus, or other enveloped or non-enveloped virus.
70 . The method of claim 68 , wherein the non-exosomal material is a recombinant protein.
71 . The method of any of claims 67-70 , wherein the isolated non-exosomal material is substantially free of exosomes.
72 . The method of any of claims 67-71 , wherein the target protein further comprises an affinity tag, wherein the affinity tag has affinity to the binding agent.
73 . The method of any of claims 67-72 , wherein the target protein comprises a polypeptide of SEQ ID NO: 33.
74 . The method of any of claims 67-73 , wherein the binding agent comprises an immunoglobulin, a protein, a peptide, or a small molecule.
75 . The method of any of claims 67-74 , wherein the binding agent is attached to a solid support, optionally wherein the solid support comprises a porous agarose bead, a microtiter plate, a magnetic bead, or a membrane.
76 . The method of claim 75 , wherein the solid support forms a chromatography column.
77 . The method of claim 76 , wherein the step of contacting the sample with the binding agent is performed by applying the sample to the chromatography column.