# US 11,421,247 B2 · Borna viral vector and use thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,421,247 B2
- Document ID: US11421247B2
- Status: Granted
- Kind: B2
- Application number: 16/493,232
- Filed: 2018-03-06
- Published: 2020-04-30
- Granted: 2022-08-23
- Earliest claimed: 2017-03-14
- Assignees: Kyoto University
- Applicants: Kyoto University
- Inventors: Keizo Tomonaga; Akiko Makino
- Web page: https://patentgrep.com/patent/US11421247B2

## CPC

- C07K 14/005: from viruses
- C12N 15/86: Viral vectors
- A61K 35/76: Viruses; Subviral particles; Bacteriophages
- A61P 25/00: Drugs for disorders of the nervous system
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 9/127: {RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase}
- C12N 2760/00022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/00041: Use of virus, viral particle or viral elements as a vector
- C12N 2760/00043: viral genome or elements thereof as genetic vector
- C12N 2760/00044: Chimeric viral vector comprising heterologous viral elements for production of another viral vector
- C12N 2760/00052: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N 2799/02: as vector
- C12N 2800/50: Vectors for producing vectors
- 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

Disclosed is a viral vector comprising (a) a cDNA of a recombinant viral RNA having a sequence of a Borna disease viral genome comprising a disrupted G gene of the Borna disease viral genome and an inserted G gene of an avian bornaviral genome, wherein the cDNA of the recombinant viral RNA has at least an N gene, an X gene, a P gene and an L gene of the Borna disease viral genome in the same order as in the Borna disease viral genome and has an inserted foreign gene; (b) DNAs encoding ribozymes; and (c) a promoter sequence, wherein (b) the DNAs encoding ribozymes are located upstream and downstream of (a) the cDNA of the recombinant viral RNA, and (a) the cDNA of the recombinant viral RNA and (b) the DNAs encoding ribozymes are located downstream of (c) the promoter sequence. The present invention can be used as a gene introduction technique that does not affect a host chromosome and can be suitable for the application in various fields, such as the treatment and prevention of brain and neurological diseases, visualization techniques of nerve cells in the field of neuroscience, etc.

## Claims

### Claim 1

A viral vector comprising a cDNA from a recombinant viral RNA having a sequence of a Borna disease viral genome comprising a disrupted G gene of from a Borna disease viral genome and an inserted G gene from an avian bornaviral genome.

### Claim 2 (depends on 1)

The viral vector according to claim 1, which comprises:
(a) the cDNA of a recombinant viral RNA having a sequence from a Borna disease viral genome comprising a disrupted G gene from the Borna disease viral genome and an inserted G gene from an avian bornaviral genome, wherein the cDNA of the recombinant viral RNA has at least an N gene, an X gene, a P gene and an L gene of the Borna disease viral genome in the same order as in the Borna disease viral genome and an inserted foreign gene;
(b) DNAs encoding ribozymes; and
(c) a promoter sequence,
wherein (b) the DNAs encoding ribozymes are located upstream and downstream of (a) the cDNA of the recombinant viral RNA, and (a) the cDNA of the recombinant viral RNA and (b) the DNAs encoding ribozymes are located downstream of (c) the promoter sequence.

### Claim 3 (depends on 2)

The viral vector according to claim 2, wherein (a) the cDNA of the recombinant viral RNA further comprises a disrupted M gene of the Borna disease viral genome.

### Claim 4 (depends on 2)

The viral vector according to claim 2, wherein (a) the cDNA of the recombinant viral RNA has the foreign gene inserted in an untranslated region between open reading frames of the P gene and the M gene of the Borna disease viral genome.

### Claim 5 (depends on 2)

The viral vector according to claim 2, wherein (c) the promoter sequence is an RNA polymerase II promoter sequence.

### Claim 6 (depends on 2)

The viral vector according to claim 2, wherein (b1) a cDNA encoding a hammer head ribozyme is located upstream of (a) the cDNA of the recombinant viral RNA, and (b2) a cDNA sequence encoding a hepatitis δ virus ribozyme is located downstream of (a) the cDNA of the recombinant viral RNA.

### Claim 7 (depends on 2)

The viral vector according to claim 2, wherein (a) the cDNA of the recombinant viral RNA comprises:
restriction enzyme sites at the 3′ and 5′ ends of the foreign gene;
a sequence of SEQ ID NO: 9 located between the restriction enzyme site at the 3′ end of the foreign gene and the open reading frame of the P gene and a sequence of SEQ ID NO: 9 located between the restriction enzyme site at the 5′ end of the foreign gene and the open reading frame of the L gene, or
when the cDNA of the recombinant viral RNA has the M gene of the Borna disease viral genome in the same location as in the Borna disease viral genome, a sequence of SEQ ID NO: 9 located between the restriction enzyme site at the 3′ end of the foreign gene and the open reading frame of the P gene and a sequence of SEQ ID NO: 9 located between the restriction enzyme site at the 5′ end of the foreign gene and the open reading frame of the M gene;
at least an inserted nucleotide sequence cc between the restriction enzyme site at the 3′ end of the foreign gene and the sequence of SEQ ID NO: 9 located near the 3′ end of the foreign gene; and
at least an inserted nucleotide sequence cca between the restriction enzyme site at the 5′ end of the foreign gene and the sequence of SEQ ID NO: 9 located near the 5′ end of the foreign gene.

### Claim 8 (depends on 1)

A recombinant virus comprising an RNA encoded by the viral vector according to claim 1.

### Claim 9 (depends on 1)

A method for preparing a recombinant virus, the method comprising:
introducing into a cell in vitro the viral vector according to claim 1 together with (i) a plasmid or plasmids expressing an N gene, a P gene and an L gene of a Borna disease viral genome and a G gene of an avian bornaviral genome or (ii) a plasmid or plasmids expressing an N gene, a P gene and an L gene of a Borna disease viral genome and a plasmid expressing a G gene of an avian bornaviral genome, as a helper plasmid or helper plasmids, and
culturing the cell having the viral vector and the helper plasmid or helper plasmids introduced therein to produce a recombinant virus.

### Claim 10 (depends on 9)

The method for preparing a recombinant virus according to claim 9, further comprising introducing a plasmid expressing an M gene of a Borna disease viral genome as a helper plasmid into the cell in vitro.

### Claim 11 (depends on 8)

A method for introducing a foreign gene, the method comprising infecting a cell or an animal with the recombinant virus according to claim 8.

### Claim 12 (depends on 11)

The method for introducing a foreign gene according to claim 11, further comprising introducing a foreign gene into cranial nerve cells.