Compositions and methods for exosome targeted expression

Abstract

The present application relates to methods of producing exosomes. The application also provides a method for preparing a protein composition comprising culturing an exosome-producing cell expressing a Nef-fusion protein comprising a Nef-derived peptide fused to a protein of interest; isolating exosomes from the exosome-producing cell culture; and purifying the protein of interest from the isolated exosomes. The application further discloses compositions that comprise exosomes containing the Nef-fusion protein, as well as methods of using the Nef-fusion protein and exosomes containing the Nef-fusion protein.

What is claimed is: 
     
         1 . A method for preparing a protein composition, comprising:
 a) culturing an exosome-producing cell expressing a Nef-fusion protein comprising a Nef-derived peptide fused to a protein of interest;   (b) isolating exosomes from cultured exosome-producing cell; and   (c) purifying said protein of interest from isolated exosomes.   
     
     
         2 . The method of  claim 1 , wherein said Nef-derived peptide comprises a Nef fragment comprising SEQ ID NO:3, or a variant of said Nef fragment. 
     
     
         3 . The method of  claim 1 , wherein said Nef-derived peptide comprises a Nef fragment comprising SEQ ID NO:4 or a variant of said Nef fragment. 
     
     
         4 . The method of  claim 1 , wherein said Nef-derived peptide comprises a Nef fragment comprising SEQ ID NO:5, or a variant of said Nef fragment. 
     
     
         5 . The method of  claim 1 , wherein said Nef-derived peptide comprises a Nef fragment comprising SEQ ID NO:6 or a variant of said Nef fragment. 
     
     
         6 . The method of  claim 1 , wherein said Nef fusion protein further comprises an amino acid sequence encoding a functional domain selected from the group consisting of affinity tag, protease cleavage site, targeting domain, reporter, enzyme and combination thereof. 
     
     
         7 . The method of  claim 1 , wherein said exosome-producing cell is stably transformed with a vector expressing said Nef-fusion protein. 
     
     
         8 . The method of  claim 1 , wherein said exosome-producing cell is transiently transfected with a vector expressing said Nef-fusion protein. 
     
     
         9 . A cell line that produces exosomes containing a Nef-fusion protein comprising a Nef-derived peptide fused to a protein of interest, wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6. 
     
     
         10 . A method for delivering a protein of interest to a target cell in a mammal, comprising administering to the mammal an exosome comprising a Nef-fusion protein, wherein said Nef-fusion protein comprises a Nef-derived peptide fused to said protein of interest and wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6. 
     
     
         11 . The method of  claim 10 , wherein said Nef-derived peptide comprises SEQ ID NO:3. 
     
     
         12 . The method of  claim 10 , wherein said Nef-derived peptide comprises SEQ ID NO:4. 
     
     
         13 . The method of  claim 10 , wherein said Nef-derived peptide comprises SEQ ID NO:5. 
     
     
         14 . The method of  claim 10 , wherein said Nef-derived peptide comprises SEQ ID NO:6. 
     
     
         15 . The method of  claim 10 , wherein said exosome is further loaded with a member from the group consisting of antigen, peptide, small molecule drug, and siRNA. 
     
     
         16 . A method for inducing an immune response in a mammal, comprising administering to a mammal an exosome comprising a Nef-fusion protein comprising a Nef-derived peptide fused to an immunogenic protein of interest, wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6. 
     
     
         17 . The method of  claim 16 , wherein said immunogenic protein comprises at least a portion of a tumor antigen. 
     
     
         18 . The method of  claim 16 , wherein said exosome is isolated from an antigen-presenting cell. 
     
     
         19 . A method for detecting a target molecule in a sample, comprising:
 contacting a sample from a subject with a Nef-fusion protein that binds specifically to said target molecule, wherein said Nef-fusion protein comprises SEQ ID NO:3, 4, 5 or 6.   detecting a binding of said target molecule in the sample to said Nef-fusion protein, and   determining a level of said target molecule in said sample,   wherein a medical condition is indicated if a level of said target molecule is outside a reference range.   
     
     
         20 . A pharmaceutical composition, comprising:
 an exosome comprising a Nef-fusion protein containing a Nef-derived peptide fused to a protein of interest; and   a pharmaceutically acceptable carrier.   
     
     
         21 . The pharmaceutical composition of  claim 20 , wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6. 
     
     
         22 . A Nef-fusion protein, produced by
 culturing cells that produce exosomes containing said Nef-fusion protein; isolating exosomes from said cells; and purifying said Nef-fusion protein from isolated exosomes, wherein said Nef-fusion protein comprises a Nef-derived peptide fused to a protein of interest and wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6.