Artificially-Manipulated Neovascularization Regulatory System

Abstract

The present invention relates to an artificially manipulated neovascularization-associated factor for regulating neovascularization and a use thereof. More particularly, the present invention relates to a system for artificially regulating neovascularization, which includes an artificially manipulated neovascularization-associated factor for regulating neovascularization and/or a composition for artificially manipulating the neovascularization-associated factor. In a specific aspect, a neovascularization regulatory system including a neovascularization-associated factor such as artificially manipulated VEGFA, HIF1A, ANGPT2, EPAS1, or ANGPTL4 and/or an expression product thereof is provided.

What is claimed is: 
     
         1 . A composition for gene manipulation, comprising:
 a guide nucleic acid capable of targeting at least one of the target sequence selected from SEQ ID NOs: 1 to 1522 in nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, or a nucleic acid sequence encoding the same; and   an editor protein or a nucleic acid sequence encoding the same.   
     
     
         2 . The composition for gene manipulation of  claim 1 , wherein the editor protein includes one or more proteins selected from the group consisting of a  Streptococcus pyogenes -derived Cas9 protein, a  Campylobacter jejuni -derived Cas9 protein, a  Streptococcus thermophilus -derived Cas9 protein, a  Streptocuccus aureus -derived Cas9 protein, a  Neisseria meningitidis -derived Cas9 protein, and a Cpf1 protein. 
     
     
         3 . The composition for gene manipulation of  claim 2 , wherein the editor protein is a  Streptococcus pyogenes -derived Cas9 protein or  Campylobacter jejuni -derived Cas9 protein. 
     
     
         4 . The composition for gene manipulation of  claim 1 , wherein the gene manipulation includes one or more modifications of nucleic acids which is
 at least one of a deletion or insertion of one or more nucleotides, a substitution with one or more nucleotides different from a wild-type gene, and an insertion of one or more foreign nucleotide, in a proto-spacer-adjacent motif (PAM) sequence in a nucleic acid sequence constituting the neovascularization-associated factor or in a continuous 1 bp to 50 bp the base sequence region adjacent to the 5′ end and/or 3′ end thereof, or   a chemical modification of one or more nucleotides in a nucleic acid sequence constituting the neovascularization-associated factor.   
     
     
         5 . The composition for gene manipulation of  claim 4 , wherein the PAM sequence includes one or more of the following sequences (described in the 5′ to 3′ direction):
 NGG (N is A, T, C or G); 
 NNNNRYAC (each N is independently A, T, C or G, R is A or G, and Y is C or T); 
 NNAGAAW (each N is independently A, T, C or G, and W is A or T); 
 NNNNGATT (each N is independently A, T, C or G); 
 NNGRR(T) (each N is independently A, T, C or G, R is A or G, and Y is C or T); and 
 TTN (N is A, T, C or G). 
 
     
     
         6 . The composition for gene manipulation of  claim 1 , wherein the composition for gene manipulation is formed in a viral vector system. 
     
     
         7 . The composition for gene manipulation of  claim 6 , wherein the viral vector includes one or more selected from a retrovirus, a lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia virus, a poxvirus and a herpes simplex virus. 
     
     
         8 . The composition for gene manipulation of  claim 1 , wherein the composition is formed in a ribonucleoprotein which is a complex of a guide nucleic acid and an editor protein, and
 wherein the guide nucleic acid is guide RNA.   
     
     
         9 . The composition for gene manipulation of  claim 1 , wherein the composition is used for treating an angiovascular disorder. 
     
     
         10 . The composition for treating of  claim 9 , wherein the angiovascular disorder is ischemic retinopathy or retinopathy of prematurity. 
     
     
         11 . A guide nucleic acid, which is capable of targeting at least one of group consisting of target sequences of SEQ ID NOs: 1 to 1522 for artificially manipulating one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene,
 wherein the target sequences of SEQ ID NOs: 1 to 1522 are in the nucleic acid sequences of the genes, and   wherein the guide nucleic acid is complexed with an editor protein for artificially manipulating the genes.   
     
     
         12 . The guide nucleic acid of  claim 11 , which includes one or more guide nucleic acids selected from the group consisting of:
 guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 3, 4, 7, 9, 10 and 11 in the nucleic acid sequence of the VEGFA gene;   guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 14, 18, 19, 20, 26, 29 and 31 in the nucleic acid sequence of the HIF1A gene;   guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 33, 34, 37, 38, 39 and 43 in the nucleic acid sequence of the ANGPT2 gene;   guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 47, 48, 49, 50, 53, 54 and 55 in the nucleic acid sequence of the EPAS1 gene; and   guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 64, 66, 67, 73, 76 and 79 in the nucleic acid sequence of the ANGPTL4 gene.   
     
     
         13 . The guide nucleic acid of  claim 11 , wherein the guide nucleic acid is a nucleotide of 18 to 23 bp. 
     
     
         14 . A method for treating an angiovascular disorder, comprising:
 introducing (administering) a composition to a subject, wherein the composition comprising:   a guide nucleic acid capable of targeting at least one of the target sequence selected from SEQ ID NOs: 1 to 1522 in nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, or a nucleic acid sequence encoding the same; and   an editor protein or a nucleic acid sequence encoding the same.   
     
     
         15 . The method of  claim 14 , wherein the angiovascular disorder is ischemic retinopathy or retinopathy of prematurity. 
     
     
         16 . The method of  claim 14 , wherein the editor protein includes one or more proteins selected from the group consisting of a  Streptococcus pyogenes -derived Cas9 protein, a  Campylobacter jejuni -derived Cas9 protein, a  Streptococcus thermophilus -derived Cas9 protein, a  Streptocuccus aureus -derived Cas9 protein, a  Neisseria meningitidis -derived Cas9 protein, and a Cpf1 protein. 
     
     
         17 . The method of  claim 14 , wherein the introducing is conducted through eyeball. 
     
     
         18 . The method of  claim 14 , wherein the composition is formed in a viral vector system. 
     
     
         19 . The method of  claim 18 , wherein the viral vector includes one or more selected from a retrovirus, a lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia virus, a poxvirus and a herpes simplex virus.