	US 11,999,952 B2
	Artificially-manipulated neovascularization regulatory system
Jeong Hun Kim, Seoul (KR); Sung Wook Park, Seoul (KR); Seokjoong Kim, Seoul (KR); and Dong Woo Song, Seoul (KR)
Assigned to TOOLGEN INCORPORATED, Seoul (KR); SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION, Seoul (KR); SEOUL NATIONAL UNIVERSITY HOSPITAL, Seoul (KR); and INSTITUTE FOR BASIC SCIENCE, Daejeon (KR)
Filed by TOOLGEN INCORPORATED, Seoul (KR); SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION, Seoul (KR); and SEOUL NATIONAL UNIVERSITY HOSPITAL, Seoul (KR)
Filed on Apr. 21, 2021, as Appl. No. 17/236,660.
Application 17/236,660 is a continuation of application No. 15/953,141, filed on Apr. 13, 2018, abandoned.
Application 15/953,141 is a continuation of application No. PCT/KR2017/009078, filed on Aug. 21, 2017.
Claims priority of provisional application 62/376,998, filed on Aug. 19, 2016.
Prior Publication US 2021/0254054 A1, Aug. 19, 2021
Int. Cl. C12N 15/00 (2006.01); A61K 9/00 (2006.01); A61K 38/46 (2006.01); A61K 48/00 (2006.01); C07K 14/47 (2006.01); C07K 14/515 (2006.01); C07K 14/52 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/113 (2010.01); C12N 15/86 (2006.01)

CPC C12N 15/11 (2013.01) [A61K 9/0048 (2013.01); A61K 38/465 (2013.01); A61K 48/00 (2013.01); A61K 48/005 (2013.01); C07K 14/4702 (2013.01); C07K 14/515 (2013.01); C07K 14/52 (2013.01); C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 15/1136 (2013.01); C12N 15/86 (2013.01); A01K 2227/105 (2013.01); A01K 2267/03 (2013.01); C12N 2310/20 (2017.05); C12N 2750/14143 (2013.01); Y02A 50/30 (2018.01)]

11 Claims

1. A method for artificially manipulating an hypoxia-inducible factor 1-alpha (HIF1A) gene in a cell, comprising introducing a composition to the cell,

in which the composition comprises

i) an editor protein or a nucleic acid encoding the editor protein, wherein the editor protein is Campylobacter jejuni Cas9 protein; and

ii) a guide nucleic acid capable of targeting a target sequence, or a nucleic acid encoding the guide nucleic acid,

wherein the target sequence is selected from SEQ ID NOs: 18, 19, 20, 26, and 29 present within the HIF1A gene on a genome in the cell and a proto-spacer-adjacent motif (PAM), wherein the PAM sequence is contiguous 8 nucleotide sequence that exists in the genome in the 3′ direction of the target sequence, which is NNNNRYAC, wherein each N is independently A, T, C or G, R is A or G, and Y is C or T,

wherein the guide nucleic acid comprises a guide domain capable of targeting a target sequence, and a sequence in which a first complementary domain, a linker domain, a second complementary domain, a proximal domain and a tail domain are linked in a direction of 5′ to 3′;

wherein the guide nucleic acid and the editor protein form a complex and induces a double strand break in nucleic acid of the HIF1A gene, and

wherein the composition induces an indel mutation by NHEJ mechanism within the HIF1A gene in the cell.