# US 6,265,183 B1 · Direct molecular cloning of foreign genes into poxviruses and methods for the preparation of recombinant proteins

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- Number: US 6,265,183 B1
- Document ID: US6265183B1
- Status: Granted
- Kind: B1
- Application number: 08/358,928
- Filed: 1994-12-19
- Granted: 2001-07-24
- Earliest claimed: 1991-08-26
- Assignees: Baxter Aktiengesellschaft
- Inventors: Friedrich Dorner; Friedrich Scheiflinger; Falko Gunter Falkner; Michael Pfleiderer
- Web page: https://patentgrep.com/patent/US6265183B1

## CPC

- C12N 15/86: Viral vectors
- A61P 31/12: Antivirals
- A61P 37/04: Immunostimulants
- C07K 14/005: from viruses
- C07K 14/745: Blood coagulation or fibrinolysis factors
- C07K 14/755: Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)}
- C12N 9/6429: {Thrombin (3.4.21.5)}
- C12N 9/6435: {Plasmin (3.4.21.7), i.e. fibrinolysin}
- C12N 9/644: {Coagulation factor IXa (3.4.21.22)}
- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- C12Y 304/21005: Thrombin (3.4.21.5)
- C12Y 304/21007: Plasmin (3.4.21.7), i.e. fibrinolysin
- C12Y 304/21022: Coagulation factor IXa (3.4.21.22)
- C12N 2710/24043: viral genome or elements thereof as genetic vector
- C12N 2710/24143: viral genome or elements thereof as genetic vector
- C12N 2740/16222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- 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
- 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)
- C12Y: ENZYMES

## Abstract

A method is disclosed for producing a modified eukaryotic cytoplasmic DNA virus by direct molecular cloning of a modified DNA molecule comprising a modified cytoplasmic DNA virus genome. The inventive method comprises the steps of (I) modifying under extracellular conditions a DNA molecule comprising a first cytoplasmic DNA virus genome to produce a modified DNA molecule comprising the modified cytoplasmic DNA virus genome; (II) introducing the modified DNA molecule into a first host cell which packages the modified DNA molecule into infectious virions; and (III) recovering from the host cell virions comprised of the modified viral genome. The host cell is infected with a helper virus which is expressed to package the modified viral genome into infectious virions. Examples of packaging a modified poxvirus genome by a helper poxvirus of the same or different genus are described. Also disclosed are novel poxvirus vectors for direct molecular cloning of open reading frames into a restriction enzyme cleavage site that is unique in the vector. In one model poxvirus vector, the open reading frame is transcribed by a promoter located in the vector DNA upstream of a multiple cloning site comprised of several unique cleavage sites.

## Claims

### Claim 1

A method for producing a protein employing a modified vaccinia viral expression system comprising the following steps:
(a) providing a modified vaccinia virus containing a heterologous insert encoding a protein, wherein said insert was molecularly cloned directly into the viral genome into a unique restriction endonuclease cleavage site;
(b) infecting a vertebrate cell culture with the modified vaccinia virus from step (a);
(c) culturing the infected cells under conditions resulting in expression of the protein; and,
(d) isolating the protein produced by the infected cells.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the protein is selected from the group consisting of HIV gp160, HIV Gag, and HIV Gag-Pol.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the protein is selected from the group consisting of prothrombin, Factor IX, Protein S, von Willebrand Factor, lys-plasminogen, and glu-plasminogen.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the restriction endonuclease is selected from the group consisting of NotI, SmaI, ApaI, and RsrII.

### Claim 5

A method for producing a protein employing a modified fowlpox viral expression system comprising the following steps:
(a) providing a modified fowlpox virus containing a heterologous insert encoding a protein, wherein said insert was molecularly cloned directly into the viral genome into a unique restriction endonuclease cleavage site recognized by a restriction endonuclease selected from the group consisting of NotI, SmaI, ApaI, and RsrII;
(b) infecting a vertebrate cell culture with the modified vaccinia virus from step (a);
(c) culturing the infected cells under conditions resulting in expression of the protein; and,
(d) isolating the protein produced by the infected cells.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the protein is selected from the group consisting of HIV gp160, HIV Gag, and HIV Gag-Pol.

### Claim 7 (depends on 5)

The method according to claim 5, wherein the protein is selected from the group consisting of prothrombin, Factor IX, Protein S, von Willebrand Factor, lys-plasminogen, and glu-plasminogen.

### Claim 8

A method for producing a protein employing a modified vaccinia viral expression system comprising the following steps:
(a) infecting cells with a modified vaccinia virus containing a heterologous insert encoding a protein, wherein said insert was molecularly cloned directly into the viral genome into a unique restriction endonuclease cleavage site;
(b) culturing the infected cells from step (a) under conditions resulting in expression of the protein.

### Claim 9 (depends on 8)

The method according to claim 8, further comprising isolating the protein produced by the infected cells of step (b).

### Claim 10 (depends on 8)

The method according to claim 8, wherein the protein is selected from the group consisting of HIV gp160, HIV Gag, and HIV Gag-Pol.

### Claim 11 (depends on 8)

The method according to claim 8, wherein the protein is a human blood protein.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the human blood protein is selected from the group consisting of prothrombin, Factor IX, Protein S, von Willebrand Factor, lys-plasminogen, and glu-plasminogen.

### Claim 13

A method for producing a protein employing a modified fowlpox viral expression system comprising the following steps:
(a) infecting cells with a modified fowlpox virus containing a heterologous insert encoding a protein, wherein said insert was molecularly cloned directly into the viral genome into a unique restriction endonuclease cleavage site recognized by a restriction endonuclease selected from the group consisting of NotI, SmaI, ApaI, and RsrII; and,
(b) culturing the infected cells from step (a) under conditions resulting in expression of the protein.

### Claim 14 (depends on 13)

The method according to claim 13, further comprising isolating the protein produced by the infected cells of step (b).

### Claim 15 (depends on 13)

The method according to claim 13, wherein the protein is selected from the group consisting of HIV gp160, HIV Gag, and HIV Gag-Pol.

### Claim 16 (depends on 13)

The method according to claim 13, wherein the protein is a human blood protein.

### Claim 17 (depends on 16)

The method according to claim 16, wherein the human blood protein is selected from the group consisting of prothrombin, Factor IX, Protein S, von Willebrand Factor, lys-plasminogen, and glu-plasminogen.