COMPOSITIONS OF ENGINEERED EXOSOMES AND METHODS OF LOADING LUMINAL EXOSOMES PAY-LOADS
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 a cargo protein.
1 . An exosome comprising a fusion protein, wherein the fusion protein comprises a biologically active payload and a scaffold protein, wherein the scaffold protein comprises MARCKS, MARCKSL1, BASP1 or a fragment or a modification thereof.
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4 . The exosome of claim 1 , wherein the exosome is produced from a cell genetically modified to comprise the exogenous sequence.
5 . The exosome of claim 4 , wherein the cell is a HEK293 cell.
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9 . The exosome of claim 1 , wherein the biologically active payload comprises a therapeutic peptide.
10 . The exosome of claim 9 , wherein the therapeutic peptide comprises a natural peptide, a recombinant peptide, or a synthetic peptide.
11 . The exosome of claim 1 , wherein the biologically active payload comprises
(i) nucleotides, amino acids, lipids, carbohydrates, or small molecules;
(ii) an antibody or a fragment of an antibody or a modification thereof;
(iii) an enzyme, a ligand, a receptor, a transcription factor, or a fragment or a modification thereof;
(iv) an antimicrobial peptide or a fragment or a modification thereof; or
(v) any combination of (i)-(iv).
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15 . The exosome of claim 1 , further comprising a second fusion protein, wherein the second fusion protein comprises MARCKS, MARCKSL1, BASP1, PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter or a fragment thereof.
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17 . The exosome of claim 1 , wherein the scaffold protein comprises a peptide expressed from a nucleotide sequence encoding the amino acid sequence (M)(G)(G/A/S)(K/Q)(L/F/S/Q)(S/A)(K)(K) (SEQ ID NO: 118).
18 . The exosome of claim 1 , wherein the scaffold protein comprises a peptide of (M)(G)(π)(X)(Φ/π)(π)(+)(+), wherein each parenthetical 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).
19 . The exosome of claim 1 , wherein the scaffold protein comprises a peptide of (M)(G)(π)(ξ)(Φ/π)(S/A/G/N)(+)(+), wherein each parenthetical 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).
20 . The exosome of claim 17 , wherein the scaffold protein comprises a peptide of any one of SEQ ID NOs: 4-110.
21 . The exosome of claim 17 , wherein the scaffold protein comprises a peptide of MGXKLSKKK, wherein X is any amino acid (SEQ ID NO: 116).
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25 . A pharmaceutical composition comprising the exosome of claim 1 and an excipient, wherein the composition is substantially free of nucleic acids, exogenous proteins, lipids, carbohydrates, metabolites, and a combination thereof.
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27 . A population of cells for producing the exosome of claim 1 , comprising an exogenous sequence encoding the scaffold protein.
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31 . The population of cells of claim 27 , wherein the exogenous sequence encodes the amino acids 1-10 of BASP1, wherein the exogenous sequence and the genomic sequence encodes the scaffold protein.
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43 . A polypeptide for modifying an exosome, comprising:
(a) a scaffold protein comprising the amino acid sequence:
(i) (M)(G)(G/A/S)(K/Q)(L/F/S/Q)(S/A)(K)(K) (SEQ ID NO: 118),
(ii) (M)(G)(ξ)(X)(Φ/π)(π)(+)(+), wherein each parenthetical 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 parenthetical 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); and
(b) a therapeutic peptide.
44 . The polypeptide of claim 43 , comprising a sequence of any of SEQ ID NO: 4-110.
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47 . The polypeptide of claim 43 , comprising a sequence of MGXKLSKKK, wherein X is any amino acid (SEQ ID NO: 116).
48 . The polypeptide of claim 43 , wherein the polypeptide is fused to a cargo peptide.
49 . The polypeptide of claim 48 , wherein the polypeptide is fused to the N-terminus of the cargo peptide.
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