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. 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 Prostaglandin F2 Receptor Negative Regulator (PTGFRN) or a fragment thereof.
2. The exosome of claim 1 , 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.
3. The exosome of claim 2 , 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.
4. The exosome of claim 1 , wherein the target protein comprises a polypeptide of SEQ ID NO: 1.
5. The exosome of claim 1 , wherein the target protein comprises a polypeptide of SEQ ID NO: 33.
6. The exosome of claim 1 , produced from a cell genetically modified to comprise the exogenous sequence, optionally wherein the cell is an HEK293 cell.
7. The exosome of claim 6 , wherein the cell is genetically modified to have a reduced expression of ADAM10.
8. The exosome of claim 6 , wherein the cell comprises a plasmid comprising the exogenous sequence.
9. The exosome of claim 6 , wherein the cell comprises the exogenous sequence inserted into a genome of the cell.
10. The exosome of claim 9 , wherein the exogenous sequence is inserted into a genomic site located 3′ or 5′ end of a genomic sequence encoding PTGFRN or a fragment thereof.
11. The exosome of claim 9 , wherein the exogenous sequence is inserted into a genomic sequence encoding PTGFRN.
12. The exosome of claim 1 , wherein the target protein is a fusion protein comprising PTGFRN or a fragment thereof, and an affinity tag, wherein the affinity tag has affinity to a binding agent.
13. The exosome of claim 1 , wherein the target protein is a fusion protein comprising PTGFRN or a fragment thereof, and a therapeutic peptide.
14. The exosome of claim 13 , 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.
15. The exosome of claim 14 , wherein the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, and small molecules.
16. The exosome of claim 14 , wherein the therapeutic peptide is an antibody or a fragment thereof.
17. The exosome of claim 14 , wherein the therapeutic peptide is an enzyme, a ligand, a receptor, or a fragment thereof.
18. The exosome of claim 1 , wherein the target protein is a fusion protein comprising PTGFRN or a fragment thereof, and a targeting moiety.
19. The exosome of claim 18 , wherein the targeting moiety is specific to an organ, a tissue, or a cell.
20. The exosome of claim 1 , further comprising a second target protein, wherein the second target protein comprises PTGFRN, BSG, IGSF3, IGSF2, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment thereof.