Compositions of Engineered Exosomes and Methods of Loading Luminal Exosome Payloads
The present invention relates to methods of preparing a therapeutic exosome using proteins newly identified to be enriched in the lumen of exosomes. Specifically, the present invention provides methods of localizing a therapeutic peptide or protein in exosomes. The methods involve generation of lumen-engineered exosomes that include one or more of the exosome proteins at higher concentrations, a modification or a fragment of the exosome protein, or a fusion protein of the exosome protein and a therapeutic or cargo 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 BASP1 or a fragment thereof.
2 . The exosome of claim 1 , wherein the target protein is present in the lumen 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 any of SEQ ID NOs: 3-15.
5 . The exosome of claim 1 , wherein the target protein comprises a polypeptide of SEQ ID NO: 14.
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 comprises a plasmid comprising the exogenous sequence.
8 . The exosome of claim 6 , wherein the cell comprises the exogenous sequence inserted into a genome of the cell.
9 . The exosome of claim 8 , wherein the exogenous sequence is inserted into a genomic site located 3′ or 5′ end of a genomic sequence encoding BASP1 or a fragment thereof.
10 . The exosome of claim 8 , wherein the exogenous sequence is inserted into a genomic sequence encoding BASP1.
11 . The exosome of claim 1 , wherein the target protein is a fusion protein comprising BASP1 or a fragment thereof, and a therapeutic peptide.
12 . The exosome of claim 11 , 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.
13 . The exosome of claim 12 , wherein the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, and small molecules.
14 . The exosome of claim 12 wherein the therapeutic peptide is an antibody or a fragment thereof.
15 . The exosome of claim 12 , wherein the therapeutic peptide is an antigen.
16 . The exosome of claim 12 , wherein the therapeutic peptide is a component of a genome editing complex.
17 . The exosome of claim 16 , wherein the genome editing complex is a CRISPR/Cas9 genome editing complex.
18 . The exosome of claim 1 , wherein the target protein is a fusion protein comprising BASP1 or a fragment thereof, and a viral capsid protein.
19 . The exosome of claim 18 , wherein the viral capsid protein is from adeno-associated virus
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.