US2020230259A1PendingUtilityA1

Isolation of extracellular vesicles (evs) from red blood cells for gene therapy

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Assignee: UNIV CITY HONG KONGPriority: Aug 16, 2017Filed: Mar 20, 2020Published: Jul 23, 2020
Est. expiryAug 16, 2037(~11.1 yrs left)· nominal 20-yr term from priority
Inventors:Thi Nguyet Minh LeJiahai ShiMuhammad Waqas
C12N 9/22A61P 35/02A61K 35/00G01N 33/491C12N 5/0641C12N 2310/20A61K 48/0075C12N 15/88C12N 2310/122A61K 35/13A61P 35/00A61K 31/7088C12N 15/907A61K 48/0041A61K 48/005C12N 2310/11A61K 48/0091A61K 35/14A61K 2035/128A61K 48/0008C12N 15/87
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Claims

Abstract

A method of RNA delivery using extracellular vesicles (EVs) derived from red blood cells (RBCs). The method comprises the purification and electroporation of the EVs and applying the RNA-loaded EVs to target cells. The method further comprises the treatment of cancer using the RNA-loaded EVs.

Claims

exact text as granted — not AI-modified
1 . A method for RNA delivery to target cells comprising:
 a) purification of extracellular vesicles (EVs) from red blood cells (RBCs);   b) electroporation of the EVs with RNAs to form RNA-loaded EVs; and   c) applying the RNA-loaded EVs to the target cells.   
     
     
         2 . The method of  claim 1 , wherein the RBCs are derived from a human and treated with calcium ionophore. 
     
     
         3 . The method of  claim 2 , wherein the EVs are purified from treated RBCs using ultracentrifugation with a sucrose cushion. 
     
     
         4 . The method of  claim 1 , wherein the RNAs comprise antisense oligonucleotides (ASO) and mRNAs. 
     
     
         5 . The method of  claim 1 , wherein the target cells comprise cancer cells. 
     
     
         6 . The method of  claim 1 , wherein the target cells comprise acute myeloid leukemia (AML) cells, breast cancer cells, or a combination of AML cells and breast cancer cells. 
     
     
         7 . The method of  claim 1 , wherein the EVs are electroporated with ASO antagonizing miR-125b. 
     
     
         8 . The method of  claim 1 , wherein the growth of the target cells is suppressed. 
     
     
         9 . The method of  claim 1 , wherein the EVs are electroporated with dextran. 
     
     
         10 . The method of  claim 1 , comprising administering to the target cells the RNA-loaded EVs which modulate an apoptosis-related gene expression, thereby inducing apoptosis in the target cells. 
     
     
         11 . A method for delivery of an antisense oligonucleotide (ASO) to target cells to suppress gene expression, wherein the method comprises:
 a) purification of extracellular vesicles (EVs) from red blood cells (RBCs);   b) electroporation of the EVs with RNAs to form RNA-loaded EVs; and   c) applying the RNA-loaded EVs to the target cells.   
     
     
         12 . The method of  claim 11 , wherein the RBCs are derived from a human and treated with calcium ionophore. 
     
     
         13 . The method of  claim 11 , wherein the RNA is an ASO antagonizing miR-125b to inhibit the oncogenic miR-125b in the target cells. 
     
     
         14 . The method of  claim 11 , wherein the target cells are acute myeloid leukemia (AML) cells, breast cancer cells, or a combination of AML cells and breast cancer cells. 
     
     
         15 . A method of RNA delivery to target cells for a CRISPR genome editing system comprising:
 a) purification of extracellular vesicles (EVs) from red blood cells (RBCs);   b) electroporation of the EVs with RNAs to form RNA-loaded EVs; and   c) applying the RNA-loaded EVs to the target cells.   
     
     
         16 . The method of  claim 15 , wherein the EVs are electroporated with Cas9 mRNA and gRNA. 
     
     
         17 . The method of  claim 15 , wherein the EVs are electroporated with Cas9 and gRNA plasmids. 
     
     
         18 . The method of  claim 15 , wherein the target cells are cancer cells. 
     
     
         19 . The method of  claim 15 , wherein the target cells are leukemia cells. 
     
     
         20 . A method of treating cancer by delivery of RNA to target cells comprising:
 a) purification of extracellular vesicles (EVs) from red blood cells (RBCs);   b) electroporation of the EVs with RNAs to form RNA-loaded EVs; and   c) applying the RNA-loaded EVs to the target cells thereby inhibiting the growth of the target cells, wherein the target cells comprise cancer cells.   
     
     
         21 . The method of  claim 20 , wherein the target cells comprises leukemia cells, breast cancer cells, or a combination of leukemia cells and breast cancer cells. 
     
     
         22 . The method of  claim 20 , wherein the target cells comprise acute myeloid leukemia cells. 
     
     
         23 . The method of  claim 20 , wherein the step c) comprises a step of administering the RNA-loaded EVs to a subject having the target cells via a local or systemic administration. 
     
     
         24 . The method of  claim 20 , wherein the growth of the target cells is suppressed after the step c).

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