Targeted and modular exosome loading system

1. An exosome comprising a packaging protein and a cargo RNA that binds to the packaging protein, wherein the packaging protein is a fusion protein comprising an RNA-binding domain and an exosome-targeting domain that targets the fusion protein to the membrane of the exosome, wherein the exosome-targeting domain is a domain of a lysosome membrane protein having a luminal N-terminus and a cytoplasmic C-terminus, and wherein the cargo RNA comprises more than one binding loop and the RNA-binding domain of the fusion protein binds specifically to the binding loops of the cargo RNA, and

further wherein the RNA-binding domain is the RNA-binding domain of MS2 bacteriophage coat protein comprising SEQ ID NO:2 or a variant thereof having at least 80% amino acid sequence identity to SEQ ID NO:2, and wherein the binding loops comprise a sequence and structure selected from the group consisting of:

where N-N is any two base-paired RNA nucleotides.

2. The exosomes of claim 1 , wherein the binding loops comprise a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or a variant thereof having at least 80% nucleotide sequence identity to SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

3. The exosomes of claim 1 , wherein the exosome-targeting domain comprises at its C-terminus a sequence selected from a group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, and SEQ ID NO:34, or a variant thereof having at least 80% amino acid sequence identity to SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, and SEQ ID NO:34, respectively.

4. The exosomes of claim 3 , wherein the exosome-targeting domain comprises at its C-terminus SEQ ID NO:24 or a variant thereof having at least 80% amino acid sequence identity to SEQ ID NO:24.

5. The exosomes of claim 4 , wherein the RNA-binding domain is fused to the C-terminus of SEQ ID NO:24 or the variant thereof.

6. The exosomes of claim 1 , wherein a ligand that targets the exosomes to target cells is present at the N-terminus of the fusion protein and is expressed on the surface of the exosomes.

7. The exosomes of claim 1 , wherein the cargo RNA is a hybrid RNA comprising the binding loops and further comprising miRNA, shRNA, mRNA, ncRNA, or a combination of any of these RNAs.

8. A method for preparing the exosomes of claim 1 , the method comprising (a) expressing in a eukaryotic cell an mRNA that encodes the fusion protein and (b) transducing into the eukaryotic cell the cargo RNA.

9. A method for delivering a cargo RNA to a target cell, the method comprising contacting the target cell with the exosomes of claim 1 .

10. A kit comprising: (a) a vector for expressing a fusion protein comprising an RNA-binding domain and an exosome-targeting domain that targets the fusion protein to the membranes of exosomes, wherein the exosome-targeting domain is a domain of a lysosome membrane protein having a luminal N-terminus and a cytoplasmic C-terminus, and (b) a vector for expressing a cargo RNA comprising more than one binding loop that bind to the RNA-binding domain of the fusion protein, wherein the RNA-binding domain is the RNA-binding domain of MS2 bacteriophage coat protein comprising SEQ ID NO:2 or a variant thereof having at least 80% amino acid sequence identity to SEQ ID NO:2, and wherein the binding loops comprise a sequence and structure selected from the group consisting of:

SEQ ID NO: 4

 U U

A   A

 N-N

 N-N

A

 N-N

 N-N

 N-N

 N-N

SEQ ID NO: 5

 C U

A   A

 N-N

 N-N

A

 N-N

 N-N

 N-N

 N-N,

and

SEQ ID NO: 6

 U C

A   A

 N-N

 N-N

A

 N-N

 N-N

 N-N

 N-N,

where N-N is any two base-paired RNA nucleotides.

11. The kit of claim 10 , wherein the vectors are separate vectors.