Artificially engineered angiogenesis regulatory system
A technology of artificial manipulation and new blood vessels, applied in the direction of angiogenin, angiogenesis factors, cardiovascular system diseases, etc., can solve the problems of no treatment for cancer metastasis or cancer, and no fundamental method for treating such diseases
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[1128] As an embodiment of the subject of the present invention, a subject containing a target gene or nucleic acid that guides the nucleic acid-editing protein complex will be described below.
[1129] Here, the target gene may be a factor related to neovascularization, such as VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene and / or ANGPTL4 gene.
[1130] The target gene can be wild-type or a modified form of the wild-type.
[1131] In an exemplary embodiment of the present invention, the subject may comprise a gene or nucleic acid manipulated by the guide nucleic acid-editing protein complex.
[1132] Here, the manipulated gene may be a factor related to neovascularization, such as VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene and / or ANGPTL4 gene.
[1133] Here, the guide nucleic acid can target neovascularization-related factors, such as VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene and / or ANGPTL4 gene.
[1134] The guide nucleic acid can be a nucleic acid sequence com...
Embodiment
[1336] Hereinafter, the present invention will be described in more detail with reference to Examples.
[1337] These examples are provided only to describe the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited to the following examples.
[1338] experimental method
[1339] 1. Design of sgRNA
[1340] Using CRISPR RGEN Tools (Institute of Basic Science, Korea), the CRISPR / Cas9 target regions of the following genes were selected: human VEGFA gene (NCBI accession number NM_001025366.2), HIF1A gene (NCBI accession number NM_001243084.1), ANGPT2 gene (NCBI accession number NM_001118887.1), EPAS1 gene (NCBI accession number NM_001430.4) and ANGPTL4 gene (NCBI accession number NM_001039667.2). Depending on the type of CRISPR enzyme, the target region of the gene can be different. The gene target sequences for CjCas9 are summarized in Table 1-Table 5 and Table 6-Table 9 listed above, and the ge...
example 1
[1430] Example 1. Confirmation of CjCas9 Expression by AAV in Mouse Retinal Tissue
[1431] Since CjCas9 consisting of 984 amino acids has a much smaller size than SpCas9 (2.95 kbp), both the CjCas9 gene and sgRNA can be packaged in one AAV vector. Therefore, in this example, in order to confirm the possibility of genetic manipulation using CjCas9 as a method for treating AMD, AAV expressing CjCas9 was used.
[1432] In order to confirm the expression of CjCas9 by AAV in mouse tissues (such as retina), under the control of U6 promoter-induced sgRNA and choroidal neovascularization (CNV)-related Vegfa gene and Hif1a gene-specific EFS promoter, construct AAV9 vector encoding CjCas9, where CjCas9 was C-terminally linked to eGFP using a self-cleaving T2A peptide (Figure 1A and Figure 1B). The constructed virus was injected into the eyeball by intravitreal injection, and after 6 weeks, CjCas9 expression in the eyeball was confirmed, indel frequency was measured using targeted de...
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