# US 2020/0399657 A1 · Improved adeno-associated virus vector

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- Number: US 2020/0399657 A1
- Document ID: US20200399657A1
- Status: Published application
- Kind: A1
- Application number: 16/968,397
- Filed: 2019-01-16
- Published: 2020-12-24
- Earliest claimed: 2018-02-07
- Assignees: Nippon Medical School Foundation; Teika Pharmaceutical Co., Ltd.
- Applicants: Nippon Medical School Foundation; Teika Pharmaceutical Co., Ltd.
- Inventors: Takashi OKADA; Tsutomu IGARASHI; Asaka SHIOZAWA
- Web page: https://patentgrep.com/patent/US20200399657A1

## CPC

- C07K 14/4753: {Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II}
- C12N 15/861: Adenoviral vectors
- A61K 35/76: Viruses; Subviral particles; Bacteriophages
- A61K 35/761: Adenovirus
- A61K 38/1833: {Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II}
- A61K 39/235: Adenoviridae
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61P 27/02: Ophthalmic agents
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/86: Viral vectors
- C07K 14/475: Growth factors; Growth regulators
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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)
- 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

## Abstract

One disadvantage of adeno-associated virus (AAV) is low gene expression efficiency due to delayed expression of the inserted gene. Provided is a vector plasmid for producing a vector genome to be inserted into an adeno-associated virus (AAV). The vector plasmid is provided with a vector genome cassette as a template of the vector genome, and the vector genome cassette is provided with: (1) an expression cassette including a nucleic acid molecule encoding a target gene and (b) a nucleic acid molecule encoding an expression regulatory region that allows expression of the target gene; and (2) two nucleic acid molecules positioned on both sides of the expression cassette and encoding inverted terminal repeats (ITRs). Also provided is a pharmaceutical composition containing the AVV vector, for treating or preventing an eye disease or disease associated with same. Further provided is a method for treating or preventing the eye disease or disease associated with same, comprising administering a therapeutically effective amount of the pharmaceutical composition for treating or preventing the eye disease or disease associated with same to a subject suffering from the eye disease or disease associated with same.

## Claims

### Claim 1

A vector plasmid for producing a vector genome to be carried in an adeno-associated virus (AAV) vector, comprising a vector genome cassette as a template of the vector genome,
wherein the vector plasmid comprises:
(1) an expression cassette comprising (a) a target gene-encoding nucleic acid molecule, and (b) an expression control region-encoding nucleic acid molecule capable of controlling expression of the target gene; and
(2) two inverted terminal repeat (ITR)-encoding nucleic acid molecules located so as to sandwich the expression cassette.

### Claim 2 (depends on 1)

The vector plasmid according to claim 1, wherein the two ITR-encoding nucleic acid molecules comprise a non-mutated ITR-encoding nucleic acid molecule and a mutated ITR-encoding nucleic acid molecule, and
the mutated ITR-encoding nucleic acid molecule has a mutation configured such that the vector genome capable of self-complementation is obtained from the vector genome cassette.

### Claim 3 (depends on 2)

The vector plasmid according to claim 2, wherein the mutated ITR-encoding nucleic acid molecule is located upstream from the non-mutated ITR-encoding nucleic acid molecule in a 5′-end direction.

### Claim 4 (depends on 1)

The vector plasmid according to claim 1, wherein the vector genome comprises the expression cassette, a reverse complementary expression cassette comprising a nucleic acid molecule encoding a reverse complementary sequence of the expression cassette, and the mutated ITR-encoding nucleic acid molecule located between the expression cassette and the reverse complementary expression cassette.

### Claim 5 (depends on 2)

The vector plasmid according to claim 2, wherein a length of the non-mutated ITR-encoding nucleic acid molecule and a length of the mutated ITR-encoding nucleic acid molecule are shorter than a length of a native ITR-encoding nucleic acid molecule thereof.

### Claim 6 (depends on 2)

The vector plasmid according to claim 2, wherein the mutated ITR-encoding nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 12.

### Claim 7 (depends on 2)

The vector plasmid according to claim 2, wherein the non-mutated ITR-encoding nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 13.

### Claim 8 (depends on 1)

The vector plasmid according to claim 1, wherein the expression control region is a truncated expression control region, and
a length of the truncated expression control region is shorter than a length of a native expression control region thereof.

### Claim 9 (depends on 8)

The vector plasmid according to claim 8, wherein the truncated expression control region has a function as a promoter.

### Claim 10 (depends on 8)

The vector plasmid according to claim 8, wherein the native expression control region is a cytomegalovirus expression control region.

### Claim 11 (depends on 10)

The vector plasmid according to claim 10, wherein the cytomegalovirus expression control region has a cytomegalovirus major immediate early promoter region.

### Claim 12 (depends on 11)

The vector plasmid according to claim 11, wherein the cytomegalovirus expression control region has a nucleotide sequence of SEQ ID NO: 1.

### Claim 13 (depends on 8)

The vector plasmid according to claim 8, wherein the truncated expression control region has a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3.

### Claim 14 (depends on 1)

The vector plasmid according to claim 1, wherein the target gene is a hepatocyte growth factor (HGF) gene.

### Claim 15 (depends on 14)

The vector plasmid according to claim 14, wherein the HGF gene is a human HGF gene.

### Claim 16 (depends on 15)

The vector plasmid according to claim 15, wherein a codon in the human HGF gene is replaced with a human frequent codon.

### Claim 17 (depends on 15)

The vector plasmid according to claim 15, wherein the human HGF gene has a nucleotide sequence of SEQ ID NO:4.

### Claim 18 (depends on 1)

An AAV vector carrying the vector genome produced from the vector plasmid according to claim 1.

### Claim 19 (depends on 18)

The AAV vector according to claim 18, for treating or preventing an eye disease or a disease associated therewith.

### Claim 20 (depends on 18)

A pharmaceutical composition for treating or preventing an eye disease or a disease associated therewith, comprising the AAV vector according to claim 18.

### Claim 21 (depends on 20)

A method for treating or preventing an eye disease or a disease associated therewith, the method comprising administering to a subject suffering from the eye disease or the disease associated therewith a therapeutically effective amount of the pharmaceutical composition for treating or preventing the eye disease or the disease associated therewith according to claim 20.