	US 11,753,621 B2
	Method for constructing functional exosomes capable of efficiently loading specific miRNA
Qiankun Li, Beijing (CN); Wenzhi Hu, Beijing (CN); Cuiping Zhang, Beijing (CN); and Xiaobing Fu, Beijing (CN)
Assigned to CHINESE PLA GENERAL HOSPITAL, Beijing (CN)
Filed by CHINESE PLA GENERAL HOSPITAL, Beijing (CN)
Filed on Nov. 8, 2021, as Appl. No. 17/520,734.
Claims priority of application No. 202011382967.2 (CN), filed on Dec. 1, 2020.
Prior Publication US 2022/0169978 A1, Jun. 2, 2022
Int. Cl. C12N 7/00 (2006.01); C12N 15/113 (2010.01); C12N 15/86 (2006.01); C12N 5/073 (2010.01); C12N 5/0775 (2010.01)

CPC C12N 5/0605 (2013.01) [C12N 5/0668 (2013.01); C12N 7/00 (2013.01); C12N 15/113 (2013.01); C12N 15/86 (2013.01); C12N 2310/141 (2013.01); C12N 2501/11 (2013.01); C12N 2501/115 (2013.01); C12N 2510/00 (2013.01); C12N 2740/15021 (2013.01); C12N 2740/15043 (2013.01)]

3 Claims

1. A method for constructing functional exosomes capable of efficiently loading specific micro-RNA (miRNA), comprising the following steps:

S1 connecting a MS2 phage capsid protein to a C1C2 domain in an exosome's Lactadherin protein to construct a C1C2-MS2 (CM) lentiviral plasmid;

S2 connecting a site pac protein to a target miRNA to construct a pac-miRNA-pac (p-miRNA-p) lentiviral plasmid, wherein the site pac protein is connected to both ends of miRNA to bind MS2;

S3 packaging the two plasmids obtained in step S1 and step S2 into lentivirus and infecting mesenchymal stem cells with the lentivirus; obtaining a confirmed stable transgenic cell line through screening of resistant drugs; saving the stem cell supernatant; and extracting the exosomes by ultracentrifugation.