# US 11,999,952 B2 · Artificially-manipulated neovascularization regulatory system

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- Number: US 11,999,952 B2
- Document ID: US11999952B2
- Status: Granted
- Kind: B2
- Application number: 17/236,660
- Filed: 2021-04-21
- Published: 2021-08-19
- Granted: 2024-06-04
- Earliest claimed: 2016-08-19
- Assignees: Toolgen Incorporated; Seoul National University R&Db Foundation; Seoul National University Hospital; Institute for Basic Science
- Applicants: Toolgen Incorporated; Seoul National University R&Db Foundation; Seoul National University Hospital
- Inventors: Jeong Hun Kim; Sung Wook Park; Seokjoong Kim; Dong Woo Song
- Web page: https://patentgrep.com/patent/US11999952B2

## CPC

- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- A61K 9/0048: {Eye, e.g. artificial tears}
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 27/02: Ophthalmic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 14/515: Angiogenesic factors; Angiogenin
- C07K 14/52: Cytokines; Lymphokines; Interferons
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/1136: {against growth factors, growth regulators, cytokines, lymphokines or hormones}
- C12N 15/86: Viral vectors
- A01K 2227/105: Murine
- A01K 2267/03: Animal model, e.g. for test or diseases
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the composition is formed in a viral vector system.

### Claim 3 (depends on 2)

The method of claim 2, wherein the viral vector includes one or more selected from a retrovirus, a lentivirus, an adenovirus, adeno-associated virus (AAV), a vaccinia virus, a poxvirus and a herpes simplex virus.

### Claim 4 (depends on 1)

The method of claim 1, wherein the composition is a ribonucleoprotein which is a complex of a guide nucleic acid and an editor protein, wherein the guide nucleic acid is a guide RNA.

### Claim 5 (depends on 2)

The method of claim 2, wherein the cell is from a subject suffering from an ocular neovascularization disease.

### Claim 6 (depends on 5)

The method of claim 5, wherein the ocular neovascularization disease is diabetic retinopathy, macular degeneration, ischemic retinopathy or retinopathy of prematurity.

### Claim 7 (depends on 1)

The method of claim 1, wherein the introducing is performed in vivo, ex vivo or in vitro.

### Claim 8

A method for treating an ocular neovascularization disease, comprising introducing or administering a composition to a subject having the ocular neovascularization disease wherein the ocular neovascularization disease is selected from the group consisting of diabetic retinopathy, macular degeneration, ischemic retinopathy, and retinopathy of prematurity,
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 which is present within a 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 the 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,
wherein the composition is formed in an adeno-associated virus (AAV) vector system, and
wherein the composition induces an indel mutation by NHEJ (Non-homologous end joining) mechanism within the HIF1A gene in the cell.

### Claim 9 (depends on 8)

The method of claim 8, wherein the AAV is AAV9.

### Claim 10 (depends on 8)

The method of claim 8, wherein the composition is in the form of a ribonucleoprotein which is a complex of a guide nucleic acid and an editor protein, wherein the guide nucleic acid is a guide RNA.

### Claim 11 (depends on 8)

The method of claim 8, wherein the composition is introduced or administered intravitreally.