# US 2012/0151614 A1 · Vector utilizing borna disease virus and use thereof

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- Number: US 2012/0151614 A1
- Document ID: US20120151614A1
- Status: Published application
- Kind: A1
- Application number: 13/258,698
- Filed: 2010-03-17
- Published: 2012-06-14
- Earliest claimed: 2009-03-31
- Assignees: Kyoto University
- Inventors: Keizo Tomonaga; Takuji Daito; Tomoyuki Honda
- Web page: https://patentgrep.com/patent/US20120151614A1

## CPC

- C12N 15/86: Viral vectors
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/121: Hammerhead
- C12N 2310/123: Hepatitis delta
- C12N 2310/128: processing or releasing ribozyme
- C12N 2330/51: Specially adapted vectors
- C12N 2760/00043: viral genome or elements thereof as genetic vector
- C12N 2760/00045: Special targeting system for viral vectors
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N 2810/6072: negative strand RNA viruses
- C12N 2810/6081: rhabdoviridae, e.g. VSV
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed is a viral vector comprising (a) a cDNA of a recombinant viral RNA having at least the N gene, the X gene, the P gene, and the L gene of a Borna disease virus genome in the same order as that in the Borna disease virus genome, and having a sequence in which a foreign gene is inserted into the untranslated region connected to the downstream of the open reading frame of the P gene, (b) a DNA encoding a ribozyme, and (c) a promoter sequence, each being disposed in a position in which (b) is placed upstream and downstream of (a), and (a) and (b) are placed downstream of (c).

## Claims

### Claim at position 1

1-14. (canceled)

### Claim 15

A viral vector comprising
(a) a cDNA of a recombinant viral RNA
having at least the N gene, the X gene, the P gene, and the L gene of a Borna disease virus genome in the same order as that in the Borna disease virus genome,
having a sequence in which a foreign gene is inserted into the untranslated region connected to the downstream of the open reading frame of the P gene,
containing restriction enzyme sites both at the 3′ end and at the 5′ end of the foreign gene,
containing the sequences shown in SEQ ID NO: 11 both in the region between the restriction enzyme site at the 3′ end of the foreign gene and the open reading frame of the P gene; and in the region between the restriction enzyme site at the 5′ end of the foreign gene and the open reading frame of the L gene, or the region between the restriction enzyme site at the 5′ end of the foreign gene and the open reading frame of the M gene when the M gene of the Borna disease viral genome is contained in the same order as that in the Borna disease viral genome, and
containing at least a base sequence cc inserted in the region between the restriction enzyme site at the 3′ end of the foreign gene and the sequence shown in SEQ ID NO: 11, and at least a base sequence cc in the region between the restriction enzyme site at the 5′ end of the foreign gene and the sequence shown in SEQ ID NO: 11
(b) a DNA encoding a ribozyme, and
(c) a promoter sequence,
each being disposed in a position in which (b) is placed upstream and downstream of (a), and (a) and (b) are placed downstream of (c).

### Claim 16 (depends on 15)

The viral vector according to claim 15, wherein (a) the cDNA of a recombinant viral RNA has a sequence in which the G gene of the Borna disease virus genome is destructed and has a sequence in which a foreign gene is inserted into the untranslated region connected to the downstream of the open reading frame of the P gene.

### Claim 17 (depends on 15)

The viral vector according to claim 15, wherein (a) the cDNA of a recombinant viral RNA has a sequence in which the G gene and the M gene of the Borna disease virus genome are destructed and has a sequence in which a foreign gene is inserted into the untranslated region connected to the downstream of the open reading frame of the P gene.

### Claim 18 (depends on 15)

The viral vector according to claim 15, wherein the restriction enzyme site at the 3′ end of the foreign gene is a Bst BI site, and the restriction enzyme site at the 5′ end of the foreign gene is a Pac I site.

### Claim 19 (depends on 15)

The viral vector according to claim 15, wherein (c) the promoter sequence is an RNA polymerase II promoter sequence.

### Claim 20 (depends on 16)

The viral vector according to claim 16, wherein an intron of the L gene of the Borna disease virus genome is deleted from (a) the cDNA of a recombinant viral RNA.

### Claim 21 (depends on 15)

A recombinant virus comprising an RNA encoded by the viral vector according to claim 15.

### Claim 22 (depends on 15)

A process for preparing a recombinant virus, the process comprising the steps of
introducing, into an in vitro cell, a plasmid or a plasmid group expressing the N gene, the P gene, and the L gene of a Borna disease virus as a helper plasmid, together with the viral vector according to claim 15, and
culturing the cell having the introduced viral vector and helper plasmid to produce a recombinant virus.

### Claim 23 (depends on 22)

The process for preparing a recombinant virus according to claim 22, wherein a plasmid expressing the coat gene of a virus is further introduced as a helper plasmid into the in vitro cell.

### Claim 24 (depends on 22)

The process for preparing a recombinant virus according to claim 22, wherein a plasmid expressing the M gene of a Borna disease virus is further introduced as a helper plasmid into the in vitro cell.

### Claim 25 (depends on 21)

A process for introducing a foreign gene, the process comprising the step of infecting an in vitro cell or an animal with the recombinant virus according to claim 21.

### Claim 26 (depends on 22)

A process for introducing a foreign gene, the process comprising the step of infecting an in vitro cell or an animal with the recombinant virus prepared by the process according to claim 22.

### Claim 27 (depends on 21)

A foreign gene transfer agent comprising the recombinant virus according to claim 21.

### Claim 28 (depends on 22)

A foreign gene transfer agent comprising a recombinant virus prepared by the process of claim 22.

### Claim 29 (depends on 21)

A foreign gene transfer agent for a cranial nerve system cell, the agent comprising the recombinant virus according to claim 21.

### Claim 30 (depends on 22)

A foreign gene transfer agent for a cranial nerve system cell, the agent comprising a recombinant virus prepared by the process according to claim 22.

### Claim 31 (depends on 15)

A kit for introducing a foreign gene, the kit comprising the viral vector according to any of claim 15.

### Claim 32 (depends on 15)

The viral vector according to claim 15, wherein the foreign gene is a DNA encoding a functional RNA.