US2020317746A1PendingUtilityA1
Targeted and modular exosome loading system
Est. expirySep 30, 2033(~7.2 yrs left)· nominal 20-yr term from priority
C12N 15/85C07K 2319/06C12N 15/88A61K 47/6901A61K 31/713A61K 9/5068C07K 2319/85C07K 14/705
58
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Claims
Abstract
Disclosed are exosomes that include a packaging protein and a cargo RNA in which the packaging protein binds specifically to the cargo RNA. The packaging protein is a fusion protein that includes an RNA-binding domain and an exosome-targeting domain. The cargo RNA includes an RNA-motif that the RNA-binding domain of the fusion protein binds specifically such that the cargo RNA is packaged in the lumen of the exosomes.
Claims
exact text as granted — not AI-modifiedWe claim:
1 . Exosomes comprising a packaging protein and a cargo RNA that binds to the packaging protein.
2 . The exosomes of claim 1 , wherein the packaging protein is a fusion protein comprising an RNA-binding domain and an exosome-targeting domain, and wherein the cargo RNA comprises an RNA-motif and the RNA-binding domain of the fusion protein binds specifically to the RNA-motif of the cargo RNA.
3 . The exosomes of claim 2 , wherein the RNA-binding domain is an RNA-binding domain of a bacteriophage, and wherein the RNA-motif is a high affinity binding loop of RNA of the bacteriophage.
4 . The exosomes of claim 3 , wherein the RNA-binding domain is the RNA-binding domain of MS2 bacteriophage 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 RNA-motif is a high affinity binding loop comprising a sequence and structure selected from the group consisting of:
U U
C U
U C
A A
A A
A A
N-N
N-N
N-N
N-N
N-N
N-N
A
A
A
N-N
N-N
N-N
N-N
N-N
N-N
N-N
N-N
N-N
SEQ ID NO: 4
N-N
SEQ ID NO: 5,
N-N
, and SEQ ID NO: 6,
N-N
where N—N is any two base-paired RNA nucleotides.
5 . The exosomes of claim 4 , wherein the high affinity binding loop comprises 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% amino acid sequence identity to SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.
6 . The exosomes of claim 3 , wherein the RNA-binding domain is the RNA-binding domain of the N-protein of lambda bacteriophage comprising SEQ ID NO:13 or a variant thereof having at least 80% amino acid sequence identity to SEQ ID NO:13, and wherein the RNA-motif is a high affinity binding loop comprising a sequence and structure selected from the group consisting of:
A
A
AG
AA
G A
G A
U-A
U-A
C-G
C-G
C-G
C-G
C-G
C-G
SEQ ID NO: 16
G-C
and SEQ ID NO: 17
GC
7 . The exosomes of claim 3 , wherein the RNA-binding domain is the RNA-binding domain of the N-protein of P22 bacteriophage comprising SEQ ID NO:14 or a variant thereof having at least 80% amino acid sequence identity to SEQ ID NO:14, and wherein the RNA-motif is a high affinity binding loop comprising a sequence and structure of:
C
A A
G A
U-A
C-G
G-C
C-G
SEQ ID NO; 18
G-C.
8 . The exosomes of claim 3 , wherein the RNA-binding domain is the RNA-binding domain of the N-protein of phi22 bacteriophage comprising SEQ ID NO:15 or a variant thereof having at least 80% amino acid sequence identity to SEQ ID NO:15, and wherein the RNA-motif is a high affinity binding loop comprising a sequence and structure of:
A A
U C
C C
U-G
C-G
C-G
A-U
C-G
U-A
SEQ ID NO: 20
U-G.
9 . The exosomes of claim 2 , wherein the exosome-targeting domain is a domain of a lysosome-associated protein.
10 . The exosomes of claim 9 , wherein the lysosome-associated protein is a lysosome membrane protein.
11 . The exosomes of claim 10 , wherein the lysosome membrane protein comprises a luminal N-terminus and a cytoplasmic C-terminus.
12 . The exosomes of claim 10 , 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.
13 . The exosomes of claim 12 , 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.
14 . The exosomes of claim 13 , wherein the RNA-binding domain is fused to the C-terminus of SEQ ID NO:24 or the variant thereof.
15 . The exosomes of claim 10 , 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.
16 . The exosomes of claim 1 , wherein the cargo RNA is a hybrid RNA comprising the RNA-motif and further comprising miRNA, shRNA, mRNA, ncRNA, or a combination of any of these RNAs.
17 . 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.
18 . A method for delivering a cargo RNA to a target cell, the method comprising contacting the target cell with the exosomes of claim 1 .
19 . A kit comprising: (a) a vector for expressing a fusion protein comprising an RNA-binding domain and an exosome-targeting domain, and (b) a vector for expressing a cargo RNA comprising an RNA-motif that binds to the RNA-binding domain of the fusion protein.
20 . The kit of claim 19 , wherein the vectors are separate vectors.Join the waitlist — get patent alerts
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