# US 2013/0156736 A1 · Complementing cell lines

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- Number: US 2013/0156736 A1
- Document ID: US20130156736A1
- Status: Published application
- Kind: A1
- Application number: 13/431,806
- Filed: 2012-03-27
- Published: 2013-06-20
- Earliest claimed: 2000-11-15
- Inventors: Ronald VOGELS; Menzo Jans Emco Havenga; Majid Mehtall
- Web page: https://patentgrep.com/patent/US20130156736A1

## CPC

- C12N 15/86: Viral vectors
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- 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 7/025: {Packaging cell lines, e.g. transcomplementing cell lines, for production of virus}
- C12N 15/861: Adenoviral vectors
- C12N 2710/10041: Use of virus, viral particle or viral elements as a vector
- C12N 2710/10051: Methods of production or purification of viral material
- C12N 2710/10343: viral genome or elements thereof as genetic vector
- C12N 2710/10352: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N 2830/00: Vector systems having a special element relevant for transcription
- C12N 2840/20: translation of more than one cistron
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

A packaging cell line that complements recombinant adenoviruses based on serotypes from subgroup B, preferably adenovirus type 35. The cell line is preferably derived from primary, diploid human cells that are transformed by adenovirus E1 sequences either operatively linked on one DNA molecule or located on two separate DNA molecules, the sequences being operatively linked to regulatory sequences enabling transcription and translation of encoded proteins. Also disclosed is a cell line derived from PER.C6 that expresses functional Ad35 E1B sequences. The Ad35-E1B sequences are driven by the E1B promoter or a heterologous promoter and terminated by a heterologous poly-adenylation signal. The cell lines are useful for producing recombinant adenoviruses designed for gene therapy and vaccination. The cell lines can also be used for producing human recombinant therapeutic proteins such as human growth factors and human antibodies. Also, the cell lines are useful for producing human viruses other than adenovirus such as influenza virus, herpes simplex virus, rotavirus, and measles virus.

## Claims

### Claim 1

An adenovirus packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, wherein said cell line comprises an adenovirus E1A coding sequence and an adenovirus E1B coding sequence each operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated into said cell line.

### Claim 2 (depends on 1)

The adenovirus packaging cell line of claim 1, wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated at different sites in said cell line.

### Claim 3 (depends on 2)

The adenovirus packaging cell line of claim 2, wherein said packaging cell line is of human origin.

### Claim 4

An adenovirus packaging cell line comprising a first expression vector and a second expression vector stably integrated into the genome of said cell line, wherein said first expression vector comprises human adenovirus E1A coding sequences, operably linked to a non-adenoviral heterologous promoter, and said second expression vector comprises human adenovirus E1B coding sequences operably linked to a non-adenoviral heterologous promoter.

### Claim 5 (depends on 1)

A method of producing the adenovirus packaging cell line of claim 1, the method comprising: introducing into a cell line permissive for adenovirus replication, nucleic acid comprising (i) an adenovirus E1A coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter and (ii) an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein the nucleic acid comprising the adenovirus E1A coding sequence and the nucleic acid comprising the adenovirus E1B coding sequence are present on separate vectors.

### Claim 6 (depends on 7)

The adenoviral vector according to claim 7, wherein said packaging cell line comprises a first expression vector and a second expression vector stably integrated into said packaging cell line's genome, wherein said first expression vector comprises adenoviral E1A coding sequences, operably linked to a non-adenoviral heterologous promoter, and said second expression vector comprises adenoviral E1B coding sequences operably linked to a non-adenoviral heterologous promoter.

### Claim 7 (depends on 1)

A method of producing an adenoviral vector substantially free of replication competent adenovirus, the method comprising: producing an andenoviral vector substantially free of replication competent adenovirus with the adenovirus packaging cell line of claim 1.

### Claim 8 (depends on 7)

The method according to claim 7, wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line.

### Claim 9 (depends on 7)

The method according to claim 7, wherein said packaging cell line is of human origin.

### Claim 10 (depends on 7)

The method according to claim 7, wherein said adenoviral vector is replication defective.

### Claim 11 (depends on 7)

The method according to claim 7, further comprising admixing the adenoviral vector substantially free of replication competent adenovirus together with a pharmaceutically acceptable excipient.

### Claim 12

A cell comprising an adenovirus E1A coding sequence and an adenovirus E1B coding sequence each operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated into said packaging cell line.

### Claim 13 (depends on 1)

The adenovirus packaging cell line of claim 1 together with an adenoviral vector substantially free of wild type replication competent adenovirus.

### Claim 14 (depends on 13)

The adenovirus packaging cell line of claim 13, wherein said adenoviral vector is replication defective.

### Claim 15 (depends on 13)

The adenovirus packaging cell line of claim 13, wherein no wild type replication competent adenovirus is detected following 18 cycles of infection.